CN1798575A - High concentration antibody and protein formulations - Google Patents

Abstract

The present application relates to highly concentrated antibody and protein formulations having reduced viscosity, stable, relatively isotonic and low turbidity. The formulations are particularly suitable for subcutaneous administration. The application further describes articles of manufacture comprising such formulations, and methods of using them to treat diseases treatable with the formulated antibodies or proteins. The formulation comprises arginine-HCl, histidine and polysorbate.

Description

High concentration antibody and protein preparation

Background technique

field of invention

The present invention relates to highly concentrated antibody formulations particularly suitable for subcutaneous administration. The invention further provides stable, highly concentrated (eg > 100 mg/ml protein) liquid formulations.

Description of related technologies

There is a significant need for highly concentrated liquid antibody preparations. However, highly concentrated protein formulations suffer from several difficulties. One difficulty is instability due to the formation of particulates. Production of liquid formulations from reconstituted lyophilized preparations has solved this problem by using surfactants (e.g., polysorbate), but surfactants are not suitable for liquid formulations because surfactants are further Processing poses difficulties. In addition, surfactants are also unable to reduce the increased viscosity, which is a result of the large number of intermolecular interactions brought about by the polymeric nature of antibodies.

Although surfactants have been shown to significantly reduce the extent of protein particle formation, they do not address the problem of increased viscosity that makes handling and administration of concentrated antibody formulations difficult. Due to the polymeric nature of antibodies and potential intermolecular interactions, antibodies tend to form viscous solutions at high concentrations. In addition, pharmaceutically acceptable sugars are often used in large quantities as stabilizers. Such sugars can enhance intermolecular interactions, thereby increasing the viscosity of the formulation. Highly viscous formulations are difficult to manufacture, draw into syringes and inject subcutaneously. The use of pressure in handling viscous formulations causes excessive foaming, which can lead to denaturation and inactivation of active biological products. This conundrum lacks a satisfactory solution.

While the prior art points to numerous examples of excipients that can be suitably used in the manufacture of pharmaceutical formulations, few proteins have been successfully formulated to concentrations greater than 100 mg/ml, and few have described the formulation of proteins to greater than 100 mg /ml concentration technique.

Applicants have found that arginine, especially arginine-HCl, is particularly suitable for highly concentrated liquid protein or antibody preparations.

Stable isotonic lyophilized protein formulations are disclosed in PCT Publication WO 97/04801, published February 13, 1997, the entire disclosure of which is incorporated herein by reference. The disclosed lyophilized formulations can be reconstituted to produce high protein concentration liquid formulations without appreciable loss of stability. However, potential problems related to high viscosity of reconstituted formulations have not been addressed. Protein aggregation has been previously reduced by the addition of sugars, but doing so significantly increases viscosity and osmotic pressure, rendering processing and use impractical.

Applicant's PCT application, WO02/30463, published April 18, 2002, discloses high protein concentration but low viscosity formulations obtained by (1) low pH (approximately 4.0 to 5.3); (2) high pH ( about 6.5 to 12.0) or (3) obtained by adding salt or buffer to increase the overall ionic strength of the formulation. However, while increased ionic strength reduces the viscosity of the formulation (eg with NaCl), it can also lead to increased solution turbidity, which is often associated with the formation of protein particles (eg, aggregation). Therefore, optimal high-concentration protein formulations must overcome the difficulties of stability, viscosity, osmotic pressure, and turbidity.

Summary of the invention

The present invention relates to highly concentrated protein or antibody formulations that are stable and are low viscosity and low turbidity.

In particular, the present invention relates to highly concentrated antibody preparations with low turbidity comprising protein or antibody (100-260 mg/ml), histidine (10-100 mM), arginine-HCl (50-200 mM) and polysorbate Alcohol esters (0.01%-0.1%) with a pH of 5.5-7.0, a viscosity of 50 cs or less and an osmotic pressure of 200-450 mOsm/kg. Alternatively, the protein or antibody in the formulation may be in the range of 120-260 mg/ml, alternatively 150-260 mg/ml, alternatively 180-260 mg/ml, alternatively 200-260 mg/ml. Or the osmolarity is in the range of 250mOsm/kg-350mOsm/kg. Alternatively, the concentration of arginine-HCl is in the range of 100-200 mM, or 150-200 mM, or 180-200 mM.

Alternatively, the invention relates to a highly concentrated antibody preparation with low turbidity, comprising antibody (40-150 mg/ml), histidine (10-100 mM), sugar (such as trehalose or sucrose, 20-350 mM) and poly Sorbitan esters (0.01%-0.1%).

In a specific embodiment, the invention provides formulations comprising high concentrations of large molecular weight proteins, such as antibodies or immunoglobulins. An antibody can be, for example, an antibody directed against a specific predetermined antigen. In a specific aspect, the antigen is IgE (eg, rhuMAbE-25 and rhuMAbE-26 described in US Patent 6,329,509 and WO99/01556). Alternatively, the anti-IgE antibody may be CGP-5101 (Hu-901) described in Corne et al., J. Clin. Invest. 99(5): 879-887 (1997), WO92/17207, and ATTC Deposit No. No. BRL-10706 and 11130, 11131, 11132, 11133. Alternatively, antigens may include: CD proteins CD3, CD4, CD8, CD19, CD20, CD34 and CD40; members of the HER receptor family such as EGF receptors, HER2, HER3 or HER4 receptors; 2C4, 4D5, PSCA, LDP -2. Cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM and αv/β3 integrins including their α- and β-subunits (eg, anti-CD11a, anti-CD18 or anti-CD11b antibody); growth factors such as VEGF; blood group antigens; flk2/flt3 receptors; obesity (OB) receptors;

The formulations of the invention may be pharmaceutical formulations. In a specific aspect, the formulation is administered subcutaneously.

In yet another embodiment, the present invention provides a method of treating, preventing or treating a disease treatable by a formulated protein or antibody comprising administering a formulation disclosed herein comprising a therapeutically effective amount of the protein or antibody. Such formulations are particularly useful for subcutaneous administration. In one aspect, the disease is an IgE-mediated disease. Further, the IgE-mediated disease is allergic rhinitis, asthma (for example, allergic asthma and non-allergic asthma), atopic dermatitis, allergic gastrointestinal disease, hypersensitivity reaction (for example, anaphylaxis, Urticaria, food allergy, etc.), allergic bronchopulmonary aspergillosis, parasitic diseases, interstitial cystitis, hyper-IgE syndrome, ataxia telangiectasia, Wiskott-Aldrich syndrome, thymic lymphoid dysplasia, IgE myeloma and graft-versus-host reaction.

In one embodiment, the invention provides an article of manufacture comprising a container containing a formulation disclosed herein. In one aspect, the article is a prefilled syringe. In another aspect, the prefilled syringe is further comprised in an injection device. In another aspect, the injection device is an auto-injector.

Description of drawings

Figure 1. Hydrophobic interaction chromatography of pepsin-digested anti-IgE monoclonal antibody. Samples were formulated in different pH values and buffers: (●) 20 mM acetate, (△) 20 mM succinate, (▲) 20 mM Na ₂ HPO ₄ , (Λ) 20 mM K ₂ PO ₄ and (*) 20 mM Tris buffer. Samples were stored at 30°C for 6 months.

Figure 2. Size exclusion chromatography of anti-IgE monoclonal antibody stored at 40°C for 6 months. Samples were prepared in different pH values and buffers: (■) 20mM glutamate, (●) 20mM acetate, (△) 20mM succinate, (□) 20mM histidine, (▲) 20mM Na ₂ HPO ₄ , () 20 mM K ₂ PO ₄ and (*) 20 mM Tris buffer.

Figure 3. Activity of anti-IgE monoclonal antibodies stored at 30°C for 6 months. Samples were prepared in different pH values and buffers: (●) 20mM acetate, (△) 20mM succinate, (□) 20mM histidine, (▲) 20mM Na ₂ HPO ₄ , () 20mM K ₂ PO ₄ and (*) 20 mM Tris buffer.

Figure 4. Effect of polysorbate 20 on turbidity of stressed anti-IgE monoclonal antibodies. Samples contained 100 mg/ml antibody, 20 mM succinate, 192 mM trehalose and various amounts of polysorbate 20, pH 6.0. The polysorbate concentrations were (■) 0, (▲) 0.01%, (●) 0.02% and (Δ) 0.05%.

Figure 5. Turbidity of anti-IgE monoclonal antibody at -150 mg/ml with different excipients (▲) _CaCl2 , ()) _MgCl2 and (Δ) Arginine-HCl.

Figure 6. Turbidity of anti-IgE monoclonal antibody at -150 mg/ml with various excipients. Samples were stored at (▲)-70°C, (■) 2-8°C, (△) 15°C, (□) 30°C and ()) 40°C.

Figure 7. Hydrophobic interaction chromatography analysis of papain digested anti-IgE monoclonal antibodies. Samples were formulated at ~150 mg/ml with various excipients and stored at () -70°C, (■) 2-8°C, (▲) 15°C, (△) 30°C and (□) 40°C.

Accompanying drawing 8. Anti-IgE monoclonal antibody is in (■) 200mM arginine-HCl, 23mM histidine, pH6.0 with～150mg/ml, (▲)182mM arginine-HCl, 20mM histidine, pH6 .0, (●) 182mM arginine-HCl, 20mM histidine, 91mM sucrose, pH6.0, (□) 50mM MgCl ₂ , 27mg/ml trehalose, 0.01% acetate, (△) 50mM MgCl ₂ , 30 mM MgAc ₂ , 0.01% acetate, and (○) 50 mM MgCl ₂ , 45 mM MgAc ₂ , 0.01% acetate. Samples were stored at 30°C for 6 months.

Figure 9. Hydrophobic interaction chromatography analysis of papain digested anti-IgE monoclonal antibodies. The samples shown were prepared in (■) 200mM Arginine-HCl, 23mM Histidine, (▲) 182mM Arginine-HCl, 20mM Histidine, (●) 182mM Arginine-HCl, 20mM Histidine, 91 mM sucrose, (□) 50 mM MgCl ₂ , 27 mg/ml trehalose, 0.01% acetate, (△) 50 mM MgCl ₂ , 30 mM MgAc ₂ , 0.01% acetate, and (○) 50 mM MgCl ₂ , 45 mM MgAc ₂ , 0.01% acetate. Samples were stored at 30°C for 6 months.

Figure 10. Shows a comparison of the full length sequences of the variable and constant chains of anti-IgE antibodies E25, E26 and Hu-901. The CDR regions of Hu-901 are underlined. For E25 and E26, the CDR regions defined by Chotbia are shown in bold, while the CDR regions defined by Kabat are drawn in boxes. Accompanying drawing 10A has shown the light chain sequence (SEQ ID NO:1-3) of E25, E26 and Hu-901, and accompanying drawing 10B has shown the heavy chain sequence of E25, E26 and Hu-901 (SEQ ID NO:4- 6).

Detailed Description of the Preferred Embodiment

I. Definition

"Protein" refers to a sequence of amino acids with chain lengths sufficient to produce high levels of tertiary and/or quaternary structure. This is the difference from "peptides" or other small molecular weight drugs that do not have this structure. Typically, the protein herein has a molecular weight of at least about 15-20 kD, preferably at least about 20 kD.

Examples of proteins encompassed by this definition are mammalian proteins, e.g., growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyrotropin; lipoproteins; alpha-1-antitrypsin ; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; ; anticoagulant factors such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen kinases such as urokinase or tissue plasminogen kinase (t-PA); bombesin; thrombin; tumors Necrosis factor-alpha and -beta; neprilysin; chemokine RANTES (regulated upon activation and normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin Such as human serum albumin; Mullerian inhibitor; relaxin A chain; relaxin B chain; pro-relaxin; mouse gonadotropin concomitant peptide; ); receptors for hormones or growth factors; integrins; proteins A or D; rheumatoid factors; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or - 6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor such as NGN-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor ( EGF); transforming growth factors (TGF) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factors-I and -II (IGF -I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD19 and CD20; (EPO); thrombopoietin (TPO); osteoinductive factors (osteoinductive factors); immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-α, -β, and -γ; CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; membrane surface proteins; decay accelerating factors (DAF); viral antigens, such as partial coats of AIDS; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; antibodies; and biologically active fragments or variants of any of the foregoing polypeptides.

The protein of the formulation is preferably substantially pure, preferably substantially homogeneous (ie, free of foreign proteins, etc.). By "substantially pure" protein is meant a composition comprising at least about 90% by weight (based on the total weight of the composition) protein, preferably at least about 95% by weight. By "substantially homogeneous" protein is meant that the composition comprises at least 99% protein by weight, based on the total weight of the composition.

In some instances, the proteins are antibodies. For example, an antibody can bind to any of the molecules described above. Typical target molecules of antibodies of the invention include CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD34 and CD40; members of the HER receptor family, such as EGF receptors, HER2, HER3 or HER4 receptors; 2c4, 4D5 , PSCA, LDP-2; cell adhesion molecules such as LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM and αv/β3 integrin, including its α or β subunits (eg , anti-CD11a, anti-CD18 or anti-CD11b antibody); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptors;

The term "antibody" as used herein includes monoclonal antibodies (including full-length antibodies with an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, bifunctional Antibodies (diabodies), and single chain molecules, and antibody fragments (eg, Fab, F(ab') ₂ , and Fv). The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody."

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units with an additional polypeptide called the J chain, containing 10 antigen-binding sites, while IgA antibodies contain 2-5 basic 4-chain units, the basic 4-chain units It can co-polymerize with J chains to form multivalent aggregates. For IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is connected to an H chain by a covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable region (V _H ) at the N-terminus, followed by three constant regions ( _CH ) for each α and γ chain, and four _CH for the μ and ε isoforms district. Each L chain has a variable region (V _L ) at the N-terminus followed by a constant region at the other terminus. The constant region of the light chain aligns with the first constant region of the heavy chain, and the variable region of the light chain aligns with the variable region of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. The pairing of _VH and _VL together forms a single antigen binding site. For the structure and properties of different classes of antibodies see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, p. 71 pages and Chapter 6.

L chains from any vertebrate species can be assigned, based on the amino acid sequence of their constant regions, into one of two distinct types, called kappa and lambda. Depending on the amino acid sequence of the constant region of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. The gamma and mu classes are further divided into subclasses based on relatively small differences in sequence and function of CH, for example, humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.

The term "variable" refers to the fact that certain segments of the variable region differ considerably in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable region. Instead, the V region consists of relatively invariant regions of about 15-30 amino acid residues separated by several shorter regions, called framework regions (FRs), with shorter regions called "Hypervariable regions," or sometimes called "complementarity determining regions" (CDRs), are of extreme variability, each about 9-12 amino acid residues in length. The variable regions of each native heavy and light chain consist of four FRs, most of which adopt a β-sheet structure, connected by three hypervariable regions, which form a circular connection and in some cases form a partial β-sheet structure. The hypervariable regions in each chain are joined together in close proximity by FRs, and together with the hypervariable regions from the other chain, form the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" (also known as "complementarity determining region" or CDR), as used herein, refers to the amino acid residues of an antibody (usually three or four hypervariable regions) located within the V region domain of an immunoglobulin. denatured short region), which forms the antigen-binding site and is the major determinant of antigen specificity. There are at least two approaches to identify CDR residues: (1) methods based on sequence variability across species (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, MS 1991)) and (2) methods based on crystallization studies of antigen-antibody complexes (Chothia, C. et al., J. Mol. Biol. 196 :901-917 (1987)). However, in the sense that the two residue calling techniques call overlapping regions, rather than identical regions, they can be combined to call hybridizing CDRs.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, a single antibody comprises a population that is identical except for minor possible natural mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant of the antigen, unlike conventional (polyclonal) antibody preparations, where there are usually different antibodies directed against different determinants (epitopes). In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized by growing hybridomas and are not contaminated by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method to produce the antibody. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method described by Kohler et al. (Nature, 256:495 (1975)), or by recombinant DNA techniques (see US Patent No. 4,816,567). "Monoclonal antibodies" can also be obtained from phage antibody libraries using techniques such as those described by Clackson et al. (Nature, 352:624-628 (1991)) and Marks et al. obtained in isolation.

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chains are combined with an antibody derived from a specific species or belonging to a specific antibody class or subclass. and the rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies are also included, as long as they Shows expected biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include "primitized" antibodies comprising variable region antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) Constant region sequence.

A "intact" antibody is one that comprises an antigen combining site as well as CL and at least the heavy chain regions _CH1 , _CH2 and _CH3 . The constant region can be a native sequence constant region (eg, a human native sequence constant region) or an amino acid sequence variant thereof. Preferably, intact antibodies have one or more effector functions.

"Antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding and/or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. [1995]); single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, termed the "Fab" fragment, and a residual "Fc" fragment, a designation that reflects the ability to readily crystallize. The Fab fragment consists of the complete L chain together with the variable region of the H chain (V _H ) and the first constant region of one heavy chain ( _CH 1 ). Each Fab fragment is monovalent for antigen binding, ie it has a single antigen binding site. Pepsin treatment of antibodies yields a single large F(ab') ₂ fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and still possesses the ability to cross-link antigen. Fab' fragments differ from Fab fragments in that Fab' has several additional residues at the carboxy-terminus of the _CH1 region, including one or more cysteines from the antibody hinge region. Fab'-SH refers here to a Fab' bearing a free thiol group at the cysteine residue in the constant domain. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portion of two H chains connected by a disulfide bond. The effector functions of antibodies are determined by the sequence of the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. In this conformation the three hypervariable domains of each variable domain interact to define an antigen-binding site on the surface of the _VH - _VL dimer. Together, these six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable region (or the half of an _FV containing only two antigen-specific hypervariable regions) has the ability to recognize and bind antigen, but with lower affinity than the full binding site.

"Single-chain Fv" may also be abbreviated as "sFv" or "scFv" antibody fragments, which include the _VH and _VL domains of an antibody, which are present on a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains, which enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small antibody fragments obtained by constructing _sFv fragments (see _previous paragraph) with a short linker (approx. The inner V regions are paired, resulting in bivalent fragments, ie, fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the _VH and _VL regions of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail in eg EP404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90 :6444-6448 (1993).

An antibody that "specifically binds" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or an epitope on a particular polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope kinds of antibodies.

The term "solid phase" describes an anhydrous matrix to which antibodies of the invention may be attached. Examples of solid phases included herein include those formed in part or in whole of glass (e.g., controlled pore size glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone resins Solid Phase. In certain embodiments, depending on the circumstances, the solid phase may comprise the wells of an assay plate; in other embodiments, it is a purification column (eg, an affinity chromatography column). The term also includes separate solid phases of intact articles, such as those described in US Patent No. 4,275,149.

"Humanized" antibodies of non-human origin (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains, or fragments (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding antibody sequences). The majority of humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the complementarity determining regions (CDRs) of the receptor are replaced by non-human ones with the desired specificity, affinity and capacity The CDR residues of an antibody such as a mouse, rat or rabbit antibody (donor antibody) are substituted. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications further refine and optimize antibody performance. The humanized antibody ideally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332 :323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2 :593-596 (1992).

A "species-dependent antibody", e.g., a mammalian anti-human IgE antibody, is one that has a higher specificity for an antigen from a first mammalian species than for a homologue of that antigen from a second mammalian species. Antibodies with stronger binding affinity. Typically, a species-dependent antibody "specifically binds" a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1 x 10 ^-7 M, or no more than 1 x 10 ^-8 M, or no more than 1 x 10 ^-9 M), but has a binding affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50-fold weaker than its affinity for said non-human antigen, At least about 500 times, or at least about 1000 times. The species-dependent antibody may be any of the various types of antibodies as defined above, but is preferably a humanized or human antibody.

Antibody "effector (organ) functions" refer to those biological activities attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (e.g., B cell receptors) downregulation of ; and B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g. natural killer (NK) cells, neutral Granulocytes and macrophages) recognize antibodies bound to target cells and subsequently cause lysis of the target cells. NK cells, the main cells that mediate ADCC, only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. For a summary of FcR expression on hematopoietic cells see Table III on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362 or US Pat. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively (or additionally), ADCC activity of the molecule of interest can be assessed in vivo, eg, in the animal model described by Clynes et al. (PNAS (USA), 95:652-656 (1998)).

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc portion of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred FcRs are receptors that bind IgG antibodies (gamma receptors), including the FcyRI, FcyRII, and FcyRIII subclasses (including allelic variants and alternatively spliced forms of these receptors). FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor") that have similar amino acid sequences, differing primarily in their cytoplasmic domains. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For reviews of FcRs, see Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Methods in Immunology 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med .126:330-41 (1995). The term "FcR" herein covers other FcRs, including those that will be identified in the future. The term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector functions. Preferably said cells express at least FcyRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMC and NK cells are preferred. The effector cells may be isolated from their natural source, eg, from blood or PBMCs as described herein.

"Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave a target in the presence of complement. Binding of the first component of the complement system (Clq) to molecules (eg, antibodies) that bind cognate antigens initiates the complement activation pathway. To assess complement activation, a CDC assay can be performed as described by Gazzano-Santoro et al. (J. Immunol. Methods 202: 163 (1996)).

An "isolated"polypeptide refers to a polypeptide that has been identified and separated and/or harvested from a component of its natural environment. Contaminating components in the peptide's natural environment are those that would interfere with the use of the peptide for diagnosis and therapy, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is purified: (1) using a rotor cup protein sequencer, to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues, or (2) using Coomassie blue Or preferably silver staining, to the extent of electrophoretic homogeneity of SDS-PAGE under non-reducing or reducing conditions. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, at least one purification step will be used to prepare isolated polypeptide.

An "isolated" nucleic acid molecule encoding the polypeptides and antibodies herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, the isolated nucleic acid is free from association with all components of the environment in which it is produced. An isolated nucleic acid molecule encoding the polypeptides and antibodies herein is in a form different from that in which it is found in its natural state or environment. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides or antibodies herein that occur naturally in cells.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

A nucleic acid is "operably linked" to another nucleic acid when the nucleic acid is in a functionally related position to the other nucleic acid sequence. For example, if a polypeptide is expressed as a precursor protein involved in the secretion of the polypeptide, the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; is operably linked to the coding sequence; or a ribosome binding site is operably linked to the coding sequence when its position facilitates translation. Generally "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading condition. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at convenient restriction sites. If no such sites exist, synthetic oligonucleotide adapters or linkers can be used according to conventional practice.

The term "epitope tagged" as used herein refers to a chimeric polypeptide comprising a polypeptide or antibody described herein fused to a "tagged polypeptide". The marker polypeptide has sufficient residues to provide an epitope against which antibodies can be raised, yet is short enough not to interfere with the activity of the polypeptide to which it is fused. The marker polypeptide is preferably also sufficiently unique that the antibody does not substantially cross-react with other epitopes. Suitable marker polypeptides generally have at least six amino acid residues, usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

The term "immunoadhesin" is defined herein as an antibody-like molecule that combines the binding domain of a heterologous "adhesin" protein (such as a receptor, ligand or enzyme) with an immunoglobulin constant region. Structurally, immunoadhesins comprise the fusion of adhesin amino acid residues having the desired binding specificity, which differ from the antigen recognition and binding site of the antibody (antigen-binding site) (that is, "heterogeneity"). The adhesin portion of an immunoadhesion molecule is typically a contiguous amino acid sequence that includes at least one binding site for a receptor or ligand. Immunoglobulin constant region sequences in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2 ), IgE, IgD or IgM. Ig fusions preferably include the replacement of at least one variable region within the Ig molecule with a region of a polypeptide or antibody as described herein. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. See also US Patent 5,428,130, issued June 27, 1995, for the production of immunoglobulin fusions.

A "stable" formulation is one in which the protein therein substantially retains its physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability exist in the art, as described in Peptide and Protein Drug Delivery, 247-301, Vincent LeeEd., Marcel Dekker, Inc. New York, New York, Pubs. (1991) and Jones A. Adv. Reviewed in Drug Delivery Rev. 10:29-90 (1993). Stability over a selected period of time can be measured at a selected temperature. For rapid screening, formulations can be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. When the formulation is to be stored at 2-8°C, generally the formulation should be stable for at least 1 month at 30°C or 40°C and/or stable for at least 2 years at 2-8°C. When the formulation is to be stored at 30°C, generally the formulation should be stable for at least 2 years at 30°C and/or at least 6 months at 40°C. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one in which less than about 10%, and preferably less than about 5%, of the protein is present as aggregates in the formulation. In another embodiment, any increase in aggregate formation during storage of the formulation can be determined.

A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent to permeate the protein. The reconstituted formulation is suitable for administration (eg, parenteral administration) to a patient in need of treatment with the protein of interest, and in certain embodiments of the invention, may be a formulation suitable for subcutaneous administration.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmolarity of from about 250 to 350 mOsm. The term "hypotonic" describes a formulation having an osmotic pressure lower than that of human blood. Accordingly, the term "hypertonic" is used to describe a formulation having an osmotic pressure higher than that of human blood. For example, isotonicity can be measured using a vapor pressure or ice-type osmometer. Formulations of the invention are hypertonic as a result of the addition of salt and/or buffer.

"Pharmaceutically acceptable acids" include inorganic and organic acids which are non-toxic in the concentrations and manner in which they are formulated. For example, suitable inorganic acids include hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfinic acid, sulfuric acid, phosphoric acid, carbonic acid, and the like. Suitable organic acids include linear and branched chain hydrocarbyl, aromatic, cyclic, cycloaliphatic, araliphatic, heterocyclic, saturated, unsaturated, monocarboxy, dicarboxy, tricarboxy, Carboxyl acids, including, for example, formic acid, acetic acid, 2-hydroxyacetic acid, trifluoroacetic acid, phenylacetic acid, trimethylacetic acid, t-butylacetic acid, anthranilic acid, propionic acid, 2-hydroxypropionic acid, 2 - Carbonylpropionic acid, malonic acid, cyclopentanepropionic acid, cyclopentane propionic acid, 3-phenylpropionic acid, butyric acid, succinic acid, benzoic acid, 3-(4- Hydroxybenzyl)benzoic acid, 2-acetoxy-benzoic acid, ascorbic acid, cinnamic acid, laurylsulfuric acid, stearic acid, muconic acid, mandelic acid, succinic acid, embenic acid, fumaric acid Acid, malic acid, maleic acid, hydroxymaleic acid, malonic acid, lactic acid, citric acid, tartaric acid, glycolic acid, sugar acid, gluconic acid, pyruvic acid, glyoxylic acid, oxalic acid, methanesulfonic acid, Succinic acid, salicylic acid, phthalic acid, palmoic acid, palmeic acid, thiocyanic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, Benzenesulfonic acid, 4-chorobenzenesulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo(2,2,2)-oct-2-ene-1-carboxylic acid, Glucoheptonic acid, 4,4'-methylenebis-3-(hydroxy-2-ene-1-carboxylic acid), hydroxynaphthoic acid.

"Pharmaceutically acceptable bases" include inorganic bases and organic bases, which are non-toxic in the concentrations and manner in which they are formulated. For example, suitable bases include those formed from inorganic alkali metals such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine bases, and organic nontoxic bases, including primary, secondary, and tertiary amines, substituted amines, cyclic amines, and base ion exchange resins, [e.g., N(R′) ⁴⁺ (where R′ is independently H or C _1-4 Hydrocarbyl, for example, ammonium, Tris)], for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine amino acid, arginine, histidine, caffeine, procaine, hypamine, choline, betaine, ethylenediamine, glucosamine, methyl glucosamine, theobromine, purine, piperazine Oxazine, piperidine, N-ethylpiperidine, polyamine resin, etc. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline and caffeine.

Additional pharmaceutically acceptable acids and bases useful in the present invention include those derived from amino acids such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine, acid and asparagine.

"Pharmaceutically acceptable"buffers and salts include those derived from the addition salts of the acids and bases specified above. Particular buffers and/or salts include histidine, succinate and acetate.

A "lyoprotectant" is a molecule that, when bound to a protein of interest, effectively prevents or reduces chemical and/or physical instability of the protein during lyophilization and subsequent storage. Exemplary lyoprotectants include sugars and their corresponding sugar alcohols; amino acids such as monosodium glutamate or histidine; methylamines such as betaine; lyotropic salts such as magnesium sulfate; or higher molecular weight sugar alcohols such as glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; Pluronics®; and its combination. Additional exemplary lysoprotectants include glycerin and gelatin, and melibiose, melezitose, raffinose, mannotriose, and stachytetose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyols selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, especially those obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. Glycoside side groups can be glucosidic or galactosidic. Further examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred lysoprotectants are the non-reducing sugars trehalose or sucrose.

Adding a lyoprotectant to a formulation prior to lyophilization by a "lyoprotectant amount" means that, after lyophilization of a protein in the presence of a lyoprotectant amount of a lyoprotectant, the protein substantially retains its Physical and chemical stability and integrity.

In preparing the reduced viscosity formulations of the present invention, care needs to be taken to utilize the above-enumerated excipients as well as other additives, especially when added in high concentrations, so as not to increase the viscosity of the formulation.

A "pharmaceutically acceptable sugar" is a molecule which, when combined with a protein of interest, effectively prevents or reduces chemical and/or physical instability of the protein during storage. A "pharmaceutically acceptable sugar" may also act as a "lysis protectant" when the formulation is to be lyophilized and then reconstituted. Exemplary sugars and their corresponding sugar alcohols include: amino acids, such as monosodium glutamate or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as tribasic or higher Sugar alcohols of molecular weight such as glycerin, dextran, erythritol, glycerol, arabinol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; Pluronics(R); and combinations thereof. Additional exemplary lysoprotectants include glycerin and gelatin, and melibiose, melezitose, raffinose, mannotriose, and stachytetose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyols selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, especially those obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. Glycoside side groups can be glucosidic or galactosidic. Further examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred pharmaceutically acceptable sugars are the non-reducing sugars trehalose or sucrose.

Addition of a pharmaceutically acceptable sugar to a formulation in a "protective amount" (e.g., prior to lyophilization) means that the protein substantially maintains its physical and chemical stability during storage (e.g., after reconstitution and storage) sex and integrity.

A "diluent" of interest here is a pharmaceutically acceptable (safe and non-toxic for administration to humans) useful agent for formulating liquid formulations such as those reconstituted after lyophilization . Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In alternative embodiments, diluents may include saline and/or buffered aqueous solutions.

A "preservative" is a compound that can be added to the formulations herein to reduce bacterial activity. Addition of preservatives may, for example, facilitate the manufacture of multiple-use (multi-dose) formulations. Examples of possible preservatives include octadecyldimethylphenylammonium chloride, hexaquaternium chloride, benzalkonium chloride (a mixture of alkylphenyldimethylammonium chlorides in which the hydrocarbyl group is a long-chain compound) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohols, hydrocarbyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexyl alcohol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

"Treatment" refers to both therapeutic treatment as well as prophylactic or preventive measures. Those in need of treatment include those already with the disease as well as those in which the disease is to be prevented.

"Mammal" for medical purposes means any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sporting or recreational animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils , mice, ferrets, house mice, cats, etc. Preferably, the mammal is a human.

A "disease" is any condition that would benefit from treatment with protein. This includes chronic and acute diseases or conditions, including those pathological conditions that predispose the mammal to the disease. Non-limiting examples of diseases in need of treatment herein include cancer and allergies.

A "therapeutically effective amount" is at least the minimum concentration required to cause measurable improvement or prevention of a particular disease. Therapeutically effective amounts of known proteins are well known in the art, and effective amounts of the proteins disclosed hereinafter can be determined by standard techniques within the purview of a skilled artisan, such as an ordinary physician.

"Viscosity" as used herein may be "kinematic viscosity" or "absolute viscosity". "Kinematic viscosity" is a measure of the resistance of a liquid to flow under the influence of gravity. When two fluids of equal volume are placed in the same capillary viscometer and allowed to flow by gravity, the viscous fluid takes longer to flow through the capillary than the less viscous fluid. If one fluid takes 200 seconds to complete its flow and the other takes 400 seconds, the second fluid is twice as viscous as the first on the scale of kinematic viscosity. "Absolute viscosity", sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density:

Absolute viscosity = kinematic viscosity × density

The unit of kinematic viscosity is L ² /T, where L is length and T is time. Typically, kinematic viscosity is expressed in centistokes (cSt). The international standard unit of kinematic viscosity is mm ² /s, which is 1cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is milliPascal-second (mPa-s), 1 cP = 1 mPa-s.

An "antihistamine" as used herein is an agent that counteracts the physiological effects of histamine. Histamine binds to its receptors, _H1 and _H2 , to produce characteristic allergic symptoms and effects, or itching, redness, swelling, etc. Many antihistamines work by blocking the binding of histamine to its receptors H1, H2; while others are thought to work by inhibiting the release of histamine. Examples of antihistamines are chlorphenamine, diphenhydramine, promethazine, cromolyn sodium, astemizole, piperheptidine maleate, bropheniramine maleate, chlorpheniramine maleate, hydrochloric acid Cetirizine, Clemastine Fumarate, Diphenhydramine Hydrochloride, Dexbrompheniramine Maleate, Dexchlorpheniramine Maleate, Chlorophylline Diphenhydramine, Diphenhydramine Hydrochloride, Succinate Duxilamine acid, fexofendadine hydrochloride, terphenadine hydrochloride, hydroxyzine hydrochloride, loratidine, minkejing hydrochloride, tripyrilamine citrate, tripyrolidine hydrochloride, propyrrolidine hydrochloride.

"Bronchodilator" as used herein describes an agent that counteracts or reverses bronchoconstriction, the physiological event that normally occurs in the early stages of an asthmatic response and leads to decreased lung capacity and panting. Examples of bronchodilators include broad acting alpha and beta adrenergic epinephrine, beta adrenergic albuterol, pirbuterol, metaproterenol, salmeterol and neoisoproterenol. Bronchodilation can also be achieved by administration of xanthines, including aminophylline and theophylline.

"Glucocorticoid" as used herein describes a steroid-based agent that has anti-inflammatory activity. Glucocorticoids are usually used to attenuate the asthmatic response in late stages. Examples of glucocorticoids include, prednisolone, beclomethasone dipropionate, fludrocortisol acetone, flunisolide, betamethasone, budesonide, dexamethasone, fludrocortisone Flunisolide, fluticasone propionate, hydrocortisone, methylprednisolone, prednisolone, prednisolone, and fludrocortisol.

"Non-steroidal anti-inflammatory drug" or "NSAID" as used herein describes a non-steroid based agent that has anti-inflammatory activity. Examples of NSAIDs include acetaminophen, acetylsalicylic acid, bromfenac sodium, diclofenac sodium, diflunisal, etodolac, phenoxyhydroatropate calcium, flurbiprofen, Ibuprofen, indole chloroformyl, ketoprofen, meclofenamate sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxybutazone, phenylbutzone, piroxicam, Linacin, sodium toluylpyramate.

II. Methods of Carrying Out the Invention

A. Peptide and Antibody Preparation

The following instructions relate primarily to the production of the polypeptides or antibodies described herein by culturing cells transformed or transfected with a vector comprising a nucleic acid encoding the same polypeptide or antibody, and purifying the protein or antibody produced. Of course, it is contemplated that alternative methods known in the art may also be used to prepare such polypeptides or antibodies. For example, such sequences, or portions thereof, can be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield , J. Am. Chem. Soc., 85 :2149-2154 (1963)]. In vitro protein synthesis can be performed using manual techniques or by automated devices. For example, automated synthesis can be achieved using an Applied Biosystems peptide synthesizer (Foster City, CA) using the manufacturer's instructions. Portions of the proteins or antibodies described herein can be chemically synthesized separately, using a combination of chemical or enzymatic methods.

1. Isolation of DNA encoding the proteins described here

DNA encoding the proteins described herein can be obtained from cDNA libraries prepared from tissues believed to possess and express the corresponding mRNA at detectable levels. Thus, such human protein-encoding DNA can be conveniently obtained from cDNA libraries prepared from human tissues, as described in the Examples. Protein-encoding genes can also be obtained from genomic libraries or by known synthetic procedures (eg, automated nucleic acid synthesis).

Libraries can be screened with probes (eg, oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest. Selected probes can be used to screen cDNA or genomic libraries using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method for isolating the gene encoding the desired gene is the PCR method [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The following examples describe techniques for screening cDNA libraries. Oligonucleotides chosen as probes should be sufficiently long and sufficiently unambiguous that false positives are minimized. The oligonucleotides are preferably labeled so as to be detectable when hybridized to DNA in the library to be screened. Methods of labeling are well known in the art and include radioactive labels such as ³² P-labeled ATP, biotinylation or enzymatic labeling. Hybridization conditions, including medium stringency and high stringency hybridization conditions, are provided in Sambrook et al., supra.

Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited in and available from public databases such as Genbank or other proprietary sequence databases. The identity (at the amino acid level or at the nucleotide level) of a sequence within a defined region of a molecule, or spanning the entire length of the sequence, can be determined using methods known in the art and as described herein.

Nucleic acids with protein-coding sequences can first be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed herein and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra. Precursor and processing intermediate of mRNA that may not be reverse transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with an expression vector or cloning vector comprising the protein or antibody to be produced as described herein, modified depending on the induction of promoters, selection of transformants, or amplification of genes encoding desired sequences cultured in conventional medium. Culture conditions, such as culture medium, temperature, pH, etc., can be selected by the skilled artisan without undue experimentation. In general, principles, protocols and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechonolgy: A Practical Approach, M. Butler, ed. (IRLPress, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to those of ordinary skill, eg, _CaCl2 , _CaPO4 , liposome-mediated methods, and electroporation methods. Depending on the host cell used, transformation is performed using standard techniques appropriate to that cell. Calcium treatment using calcium chloride as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used to transform certain plant cells as described by Shaw et al., Gene, 23 :315 (1983) and WO 89/05859, published June 29, 1989. For mammalian cells without cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52 :456-457 (1978) can be used. General aspects of transfection of mammalian cell host systems have been described in US Patent No. 4,399,216. Transformation into yeast is generally carried out according to the method of Van Solingen et al., J.Bact., 130 : 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) 76 : 3829 (1979). However, other methods for introducing DNA into cells can also be used, such as nuclear microinjection, electroporation, fusion with intact cell bacterial protoplasts, or methods of polycations such as Polybrene, polyornithine. Various techniques for transforming mammalian cells are described in Keown et al., Methods in Enzymology, 185 :527-537 (1990) and Mansour et al., Nature, 336 :348-352 (1988).

Suitable host cells for cloning or expressing the DNA herein in a vector include prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, eg, Enterobacteriaceae, such as Escherichia coli. Various strains of Escherichia coli are publicly available, for example Escherichia coli K12 strain MM294 (ATCC 31,446); Escherichia coli X1776 (ATCC 31,537); Escherichia coli strain W3110 (ATCC 27, 325) and K5772 (ATCC 53, 635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella ), Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella ( Shigella), and Bacillus species such as B. subtilis and B. licheniformis (for example, B. licheniformis 41P disclosed in DD 266,710 published on April 12, 1989), Pseudomonas, such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is a particularly preferred host or parental host because it is a universal host strain for the fermentation of recombinant DNA products. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to cause genetic mutations in genes encoding proteins endogenous to the host, examples of such hosts include Escherichia coli W3110 strain 1A2, which has the complete genotype tonA; Escherichia coli W3110 strain 9E4, which has the complete genotype tonA ptr3; Escherichia coli W3110 strain 27C7 (ATCC 55, 244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT kan ^r ; Escherichia coli Escherichia coli W3110 strain 37D6, which has the complete genotype tonAptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG kan ^r ; Escherichia coli W3110 strain 40B4, which is a non-kanamycin-resistant degP deletion mutation strain 37D6; and Escherichia coli strains with specific periplasmic proteases disclosed in US Patent No. 4,946,783, issued August 7,1990. Alternatively, in vitro cloning methods, eg, PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable hosts for cloning or expression of vectors encoding FGF-19. Saccharomyces cerevisiae, or commonly baker's yeast, is most commonly used among lower eukaryotic host microorganisms. Other eukaryotic microorganisms include: Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 [1981]; EP 139383 published May 2, 1985); Kluyveromyces host (US Patent 4943529; Fleer et al., Bio/Technologv, 9:968-975 (1991)), for example Kluyveromyces lactis (K.lactis) (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), Kluyveromyces spp. K. wickeraml] (ATCC24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC36906; Van den Berg et al., Bio/Technology, 8:135 (1990)) , heat-resistant Kluyveromyces (K.thermotolerans) and Kluyveromyces (K.marxianus), etc.; yarrowia (EP 402226); Pichia pastoris (EP 183070); .Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244234); Neurospora crassa (Case etc., Proc.Natl.Acad.Sci.USA, 76:5259-5263[ 1979]); Schwanniomyces (schwanniomyces) such as Western Schwanniomyces occidentalis (EP 394538 published on October 31, 1990); and filamentous fungi, such as Neurospora, Penicillium , Tolypocladium (WO 91/00357 published on January 10, 1991) and Aspergillus hosts such as Aspergillus nidulans (Ballance et al., Biochem.Biophys.Res.Commun., 112:284289 [1983]; Tilburn et al., Gene, 26 :205-221[1983]; Yelton et al., Proc.Natl.Acad.Sci.USA, 81:1470-1474[1984]) and Aspergillus niger et al. (Kelly and Hynes, EMBO J., 4:475-479[1985 ]). Methylotropic yeasts are suitable herein, including but not limited to yeasts capable of growing in methanol selected from the genera Hansenula, Candida, Koehler Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula. An illustrative list of specific Saccharomyces genera of such yeasts is found in C. Anthony, The Biochemistrvo of Methylotrophs, 269 (1982).

Suitable host cells for expressing glycosylated forms of the polypeptides and antibodies described herein are derived from multicellular organisms. Examples of invertebrate cells include insect cells (eg Drosophila S2 and Spodoptera Sf9) and plant cells. Useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or subclones cultured to 293 cells grown in suspension culture, Graham et al. J.Gen Virol.36:59(1977)); Chinese hamster ovary cell/-DHFR (CHO, Urlaub and Chasin, Proc.Natl.Acad.Sci.USA, 77:4216(1980)); Rat Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and murine breast cancer cells (MMT 060562, ATCC CCL51). Selection of suitable host cells is common knowledge in the art.

3. Selection and application of replicative vectors

Nucleic acids (eg, cDNA or genomic DNA) encoding polypeptides or antibodies described herein can be inserted into replicable vectors for cloning (DNA amplification) or expression. Various vectors are available to the public. A vector may be in the form of, for example, a plasmid, cosmid, viral particle or phage. Suitable nucleic acid sequences can be inserted into vectors using a variety of methods. Typically, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Suitable vectors comprising one or more of these components are constructed using standard ligation techniques well known to those skilled in the art.

Recombinant products of polypeptides or antibodies can not only be directly recombinantly prepared, but also can be prepared as fusion polypeptides with heterologous polypeptides, which are signal sequences or have specific cleavage sites on the N-terminus of mature proteins or polypeptides. other peptides. Typically, the signal sequence may be a component of the vector, or may be part of the DNA encoding the polypeptide or antibody inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from eg alkaline phosphatase, penicillinase, 1pp or thermostable enterotoxin II leader. For yeast secretion, it can be, for example, the yeast invertase leader, the alpha factor leader (including the alpha factor leaders of Saccharomyces and Kluyveromyces, the latter being described in US Patent 5010182), or the acid phosphatase leader region, the Candida albicans glucoamylase leader (EP 362179 published on April 4, 1990), or the signal sequence described in WO90/13646 published on November 15, 1990. In the case of expression in mammalian cells, mammalian signal sequences, such as signal sequences from secreted polypeptides from the same or related species, and viral secretory leaders may be used for direct secretion of the protein.

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Such sequences are well known in a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin of replication is suitable for yeast, and various viral origins of replication (SV40, polyoma, adenovirus, VSV or BPV) are suitable for mammalian cells cloning vector in .

Expression and cloning vectors often contain selectable genes, also known as selectable markers. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) compensate for auxotrophs, or (c) provide Key nutrients not available in the base, such as the gene encoding D-alanine racemase in Bacillus sp.

Examples of suitable selection markers for mammalian cells are those selectable markers that allow identification of the cells, eg DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is a DHFR activity-deficient Chinese hamster ovary (CHO) cell prepared and propagated according to the method described by Urlaub et al. in Pric. Tie. A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for yeast mutants that cannot grow in tryptophan (eg, ATCC 44076 or PEP4-1) [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors generally contain a promoter operably linked to the DNA sequence to direct mRNA synthesis. Promoters recognized by various potential host cells are well known. Promoters suitable for prokaryotic hosts, including β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, Tryptophan (trp) promoter system [Goeddel, Nucleic acids Res., 8:4057 (1980); EP 36776], and hybrid promoters such as tac promoter [deBoer et al., Proc.Natl.Acad.Sci.USA , 80:21-25 (1983)]. Promoters suitable for use in bacterial systems also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA sequence.

Examples of suitable promoter sequences for yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg. , 7:149 (1968); Holl and Biochemistrv, 17:4900 (1978)], the promoters of other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, acetone Acid decarboxylase, phosphofructokinase, glucose-6 phosphate isomerase, glycerol-3-phosphate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters, those inducible promoters that have the advantage of controlling transcription by growth conditions, are the promoter regions of genes such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, and nitrogen metabolism Associated degradative enzymes, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression systems are further described in EP 73657.

In mammalian host cells, the transcription of the vector can be regulated by a promoter derived from a viral genome such as polyoma virus (polyoma virus), fowlpox virus (fowlpox virus) (published on July 5, 1989 UK 2211504), adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), or from heterogeneous Promoters of mammalian origin, such as actin promoters or immunoglobulin promoters, etc., and promoters from heat shock, provided these promoters are compatible with the host cell system.

Transcription of nucleic acids encoding polypeptides or antibodies herein by higher eukaryotes can be increased by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA that act on promoters to increase transcription, generally about 10 to 300 bp. Enhancer sequences are known for many mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers of eukaryotic viruses are commonly used. Examples include the SV40 enhancer (bp 100-270) on the late side of its origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of its replication origin, and the adenovirus enhancer. The enhancer may be spliced into the vector 5' or 3' to the coding sequence, but is preferably located 5' to the promoter.

Expression vectors for eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) also include sequences necessary for transcription termination and stabilization of the mRNA structure. These sequences are usually derived from the 5' (occasionally 3') untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions comprise stretches of nucleotides that are transcribed as polyadenylated fragments in the untranslated region of an mRNA encoding a polypeptide or antibody described herein.

For other suitable methods, vectors and host cells suitable for the synthesis of the polypeptides or antibodies described herein in recombinant vertebrate cell culture, see Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40- 46 (1979); described in EP 117060 and EP 117058.

4. Detection of gene amplification/expression

Amplification and/or expression of a gene can be determined directly in a sample, e.g., by conventional techniques such as Southern blotting, Northern blotting to measure mRNA transcript levels, based on the sequences provided herein and using appropriately labeled probes [Thomas, Proc. USA, 77:5201-5205 (1980)], dot blot (DNA analysis) or in situ hybridization, gene amplification and/or expression can be determined directly in the sample. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, are used. Antibodies are sequentially labeled to analyze which part of the duplex is bound to the surface, so that antibodies bound to the duplex are detected based on duplex formation on the surface.

Alternatively, for direct quantification of expressed gene products, gene expression is measured using immunological methods, such as immunohistochemical staining of cells or tissue sections and analysis of cell cultures or body fluids. Antibodies suitable for immunohistochemical staining and/or sample fluid analysis can be monoclonal antibodies or polyclonal antibodies, and antibodies can be prepared in any mammal. Conveniently, peptides may be directed against the polypeptides described herein, or against peptides synthesized from the DNA sequences provided herein, or against exogenous sequences fused to DNA encoding such polypeptides and antibodies, and against peptides encoding specific antibody epitopes. Foreign sequences fused to DNA to produce antibodies.

5. Peptide purification

Polypeptides can be recovered from culture medium or host cell lysates. If the polypeptide is membrane bound, it is released from the cell membrane using a suitable detergent solution (eg Triton-X 100) or by enzymatic cleavage. Cells used to express a polypeptide or antibody described herein are disrupted using various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption or cell lysing agents.

It may be desirable to purify the polypeptides or antibodies described herein from recombinant cellular proteins or other polypeptides. The following procedures are illustrative of suitable purification steps: fractionation on ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or cation-exchange resins such as DEAE; Precipitation; gel filtration using eg Sephadex G-75; protein A Sepharose column to remove contaminants such as IgG; and metal chelator column that binds epitope-tagged form of FGF-19. Various methods of protein purification can be used and are well known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982) described. The choice of purification steps depends, for example, on the nature of the production method and the particular polypeptide or antibody produced.

B. Antibody Preparation

In certain embodiments of the invention, the selected protein is an antibody. The following are techniques for producing antibodies, including polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

(1) Polyclonal antibody.

Polyclonal antibodies are typically elicited in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Utilize bifunctional or derivatizing reagents such as maleimide benzoyl succinimidyl sulfate (binding via cysteine residues), N-hydroxysuccinimide (via lysine residues), Glutaraldehyde, succinic anhydride, _SOCl2 , or ^R1N =C=NR, where R and ^R1 are independently lower hydrocarbyl groups, binds the relevant antigen to a protein that is immunogenic in the species being immunized , are useful, such proteins as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's incomplete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic dicorynomycolate). Immunization regimens can be selected by those skilled in the art without undue experimentation.

Animals are immunized with the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbit or mouse, respectively) with 3 volumes of Freund's complete adjuvant , achieved by intradermally injecting the solution at multiple locations. One month later, animals are boosted by subcutaneous injection at multiple sites with 1/5 to 1/10 the initial amount of peptide or conjugate in Freund's complete adjuvant. Seven to fourteen days later, the animals are bled and the serum is analyzed for antibody titers. Animals were boosted until titers reached peak levels. Conjugates can also be produced in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum are suitable for enhancing the immune response.

(2) Monoclonal antibody.

Monoclonal antibodies are antibodies obtained from a population of substantially homogeneous antibodies, i.e., containing, except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts, Each antibody of the population is identical. Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of different antibodies.

For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler et al., Nature, 256 :495 (1975), or by recombinant DNA methods (US Patent No. 4,816,567).

In the hybridoma approach, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be sensitized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

The immunizing agent generally includes an antigenic protein or a fusion variant thereof. In general, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, and spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. Lymphocytes are then fused with an immortal cell line using a suitable fusion reagent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103] .

Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Typically rat or mouse myeloma cell lines are used. Hybridoma cells may be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), and the medium for hybridomas usually includes hypoxanthine, aminopterin, and thymidine ("HAT medium"), which inhibit HGPRT. - Defective cell growth.

Preferred immortal myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium, such as HAT medium. Among these, preferred are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, California USA, and those available from American Type SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) obtained from the Culture Collection, Manassus, Virginia USA. The use of human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies has also been described ((Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp.51-63 (Marcel Dekker Inc., New York, 1987)).

The medium in which the hybridoma cells were grown was analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The medium in which the hybridoma cells are grown can be analyzed for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and analytical methods are known in the art. Binding affinity can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

After identification of hybridoma cells producing antibodies of the desired specificity, affinity and/or activity, the clones are subcloned by limiting dilution steps and cultured by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can be grown in vivo, such as in mammalian ascites tumors.

Monoclonal antibodies secreted by the subclones are suitably combined with culture medium, peritoneal Effusion or serum separation.

Monoclonal antibodies can also be produced by recombinant DNA methods, eg, in US Patent No. 4,816,567 and as described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that bind specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then used to transfect host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, For the synthesis of monoclonal antibodies in recombinant host cells. Reviews of recombinant expression in bacteria of antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130 :151-188 (1992).

In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348 :552-554 (1990). Clackson et al., Nature, 352 :624-628 (1991) and Marks et al., J. Mol. Biol., 222 :581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications described strategies for the construction of large phage libraries by strand shuffling (strain shuffle) (Marks et al., Biol/Technology, 10 :779-783 (1992)) as well as combinatorial infection and in vivo recombination (Waterhouse et al., Nucl. acids Res., 21 :2265-2266 (1993)), to produce high affinity (nM range) human antibodies. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

For example, it can also be achieved by substituting the coding sequences for the human heavy and light chain constant regions for the homologous murine sequences (US Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81 :6851 (1984)) , or modify the DNA by covalently linking an immunoglobulin coding sequence to all or part of a non-immunoglobulin polypeptide coding sequence. Typically such non-immunoglobulin polypeptides replace the constant region of the antibody, or can replace the variable region of one of the antibody's antigen-binding sites, to generate chimeric bivalent antibodies comprising One antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

The monoclonal antibodies described herein may be monovalent, and methods for their preparation are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. Typically the heavy chain is truncated at any point in the Fc region to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residue can be replaced by another amino acid residue, or deleted, to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those utilizing cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

(3) Humanized antibody.

The antibodies of the present invention may further include humanized antibodies or human antibodies. Non-human (e.g., murine) humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding subsequences of antibodies ), which contain very little non-human immunoglobulin sequence. Humanized antibodies include antibodies from non-human species such as mice, rats, and rabbits in which the recipient complementarity-determining region (CDR) residues in human immunoglobulins (recipient antibodies) have been modified to have the desired specificity, affinity, and performance (donor antibody) CDR residues were substituted. In some instances, Fv framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the donor CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and usually both, variable domains, wherein all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FR sequences are Human immunoglobulin consensus sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Methods of humanizing non-human antibodies are known to those skilled in the art. Typically, one or more amino acid residues of non-human origin have been introduced into a humanized antibody. These non-human amino acid residues are often referred to as "imported" residues, which are usually from an "imported" variable region. The humanization process is basically the same as that of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 ( 1988)), by substituting one or more CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (US Patent 4816567) in which a substantial portion of an intact human variable region has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous positions in rodent antibodies.

The selection of human variable regions, including light and heavy chain variable regions, in making humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" approach, the variable region sequences of rodent antibodies are screened against the complete library of known human variable region sequences. The human sequences closest to the rodent sequence were then used as the human framework (FR) of the humanized antibody. Sims et al., J. Immunol. 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196 :901 (1987). Another approach uses a specific framework derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chains. The same backbone can be used for several different humanized antibodies. Carter et al, Proc. Natl. Acad. Sci. USA , 89 :4285 (1992);

More importantly, after the antibody is humanized, it still maintains a high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, ie, the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, CDR residues directly and most substantially affect antigen binding.

Various forms of humanized antibodies are contemplated by the present invention. For example, a humanized antibody can be an antibody fragment, such as a Fab, that is selectively conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, a humanized antibody can be a whole antibody, such as a whole IgG1 antibody.

(4) Human Antibody

As an alternative to humanization, human antibodies can be produced. For example, transgenic animals (eg, mice) can be prepared that, upon immunization, produce a repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been shown that in chimeric and germ-line mutant mice, homozygous deletion of the antibody heavy chain joining region ( _JH ) gene results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays to this germline mutant mouse will result in the production of human antibodies against antigenic challenge. See, eg, Jakobovits et al., Proceedings of the National Academy of Sciences USA 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); US Patent No. 5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce human antibodies or antibody fragments in vitro from immunoglobulin variable (V) region gene repertoires from unimmunized donors. McCafferty et al., Nature 348 :552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227 :381 (1991). According to this technique, antibody V region genes are cloned into the reading frame of the major and minor coat protein genes of filamentous phage, such as M13 or fd, which are presented as functional antibody fragments on the surface of the phage particle . Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection for the functional properties of the antibody also generates selection for the gene encoding the antibody exhibiting those properties. Thus, phages mimic some properties of B cells. Phage display can be performed in various formats, as reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3 :564-571 (1993). V gene fragments from several sources can be used for phage display. Clackson et al., Nature 352 :624-628 (1991) isolated an array of various anti-azolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V genes from unimmunized human donors can be constructed following the techniques described in Marks et al., J.Mol.Biol. 222 :581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993) Antibodies against various antigens (including autoantibodies) can be isolated substantially. See, US Patent Nos. 5,565,332 and 5,573,905.

The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147 ( 1): 86-95 (1991). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice, in which endogenous immunoglobulin genes have been Partially or completely inactivated. Upon challenge, human antibody production was observed, which was similar in all respects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. For example, 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10 :779-783 (1992); Lonberg et al. Nature 368 :856-859 (1994); Morrison, Nature 368 :812-13 (1994), Fishwild et al, Nature Biotechnology 14 :845-51 (1996), Neuberger, Nature Biotechnology 14 :826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol . 13 :65-93 (1995) described.

Finally, human antibodies can also be produced in vitro by activated B cells (see US Patent Nos. 5,567,610 and 5,229,275).

(5) Antibody fragments

In some cases, it is advantageous to use antibody fragments rather than whole antibodies. Smaller fragment sizes allow for rapid clearance, which can result in increased exposure to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem Biophys. Method. 24 :107-117 (1992); and Brennan et al., Science 229 :81 (1985) ). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large quantities of these fragments. Antibody fragments can be isolated from antibody phage libraries as described above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically ligated to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10 :163-167 (1992)). According to another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') ₂ with increased in vivo half-life are described in US Patent No. 5,869,046. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US Patent No. 5,571,894 and US Patent No. 5,587,458. Antibody fragments can also be "linear antibodies," eg, as described in US Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

(6) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

Antibodies of the invention can also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptide-based chemotherapeutic, see WO 81/01145) into an active of anticancer drugs. See, e.g., WO 88/07378 and U.S. Patent No. 4,975,278.

The enzyme component of the immunoconjugates useful for ADEPT includes any enzyme that acts on the prodrug in a manner to convert it to its more active, cytotoxic form.

Enzymes useful for the methods of the invention include, but are not limited to, glycosidase, glucose oxidase, human lysozyme, human glucuronidase, alkaline phosphatase useful for converting phosphate-containing prodrugs to free drugs; Arylsulfatase enzymes useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases useful for converting non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases such as Serratia proteases, thermolysins, subtilisins, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A) and cathepsins (e.g., cathepsins B and L), for precursors that will contain peptides D-alanyl carboxypeptidase, useful for converting prodrugs containing D-amino acid substituents; carbohydrate lyase, for example for converting glycosylated prodrugs β-galactosidase and neuraminidase useful for free drug; β-lactamase useful for converting β-lactam derived drug into free drug; and penicillin amidase, such as penicillin V amidase or Penicillin G amidase, is useful for converting drugs derivatized on the amine nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, to the free drug. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328 :457-458( 1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of abzymes to tumor cell populations.

The enzymes described above can be covalently linked to the polypeptides or antibodies described herein by techniques well known in the art, eg, using the heterobifunctional cross-linking reagents described above. Alternatively, fusion proteins comprising at least the antigen-binding region of an antibody of the invention linked to at least one functionally active portion of an enzyme of the invention can be prepared using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al. , Nature 312 :604-608 (1984)).

7) Bispecific and multispecific antibodies

Bispecific antibodies (BsAbs) are those antibodies that have binding specificities for at least two different epitopes, including those on the same or another protein. Alternatively, one arm can be equipped to bind the antigen of interest, and the other arm can be compounded with an arm that binds to a triggering molecule on a leukocyte, such as a T cell receptor molecule (eg, CD3) or IgG. Fc receptors (FcγRs), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), to focus and localize cellular defense mechanisms on cells expressing target antigens. Such antibodies may be derived from full length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies).

Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the antigen of interest. Such an antibody possesses one arm that binds to the desired antigen and another arm that binds to a cytotoxic agent (for example, saporin, anti-interferon-alpha, vinca alkoloid, ricin A chain, methotrexate, or radioisotope moiety antigen). Examples of known bispecific antibodies include anti-ErbB2/anti-FcgIII (WO 96/16673), anti-ErbB2/anti-FcgRI (US Patent 5,837,234), anti-ErbB2/anti-CD3 (US Patent 5,821,337).

Methods of making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. Millstein et al., Nature, 305 :537-539 (1983). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually by an affinity chromatography step, is rather tedious and the product yields are low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J., 10 :3655-3659 (1991).

According to a different approach, antibody variable regions with the desired binding specificity (antibody-antigen combining site) are fused to immunoglobulin constant region sequences. The fusion is preferably to an immunoglobulin heavy chain constant region, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have a first heavy chain constant region (CH1) comprising the site necessary for light chain association, present in at least one of said fusions. DNA encoding the immunoglobulin heavy chain fusion, and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows, in embodiments, great flexibility in adjusting the relative ratios of the three polypeptide fragments when unequal ratios of the three polypeptide chains provide for optimum yield when used in construction. However, when expression of at least two polypeptide chains in equal ratios results in high yields, or when said ratios are of no particular significance, it is possible to insert two or all three polypeptide chain coding sequences into one expression vector.

In a preferred embodiment of this method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with the first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain on the other arm Pair (provides a second binding specificity) composition. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain conjugates, the presence of immunoglobulin light chains in only half of the bispecific molecule providing easy separation route. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology 121 :210 (1986).

According to another approach described in WO 96/27011 or US Pat. No. 5,731,168, the interface between pairs of antibody molecules can be designed to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferably the interface comprises at least a portion of the CH3 region of the antibody constant region. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). By replacing large amino acid side chains with small ones (eg, alanine or threonine), compensatory "cavities" of the same or similar size as the large side chains are created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products, such as homodimers.

Methods for generating bispecific antibodies from antibody fragments have been described in the literature. For example, chemical linkage can be used to prepare bispecific antibodies. Brennan et al., Science 229 :81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab') ₂ fragments. These fragments were reduced in the presence of the dimercapto complexing agent sodium arsenite to stabilize adjacent dimercaptos and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to trinitrobenzene (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back into a Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibodies can be used as agents for the selective immobilization of enzymes.

Fab' fragments can be recovered directly from E. coli and chemically linked to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175 :217-225 (1992) describe the production of fully humanized bispecific antibody F(ab') ₂ molecules. Each Fab' fragment was secreted separately from E. coli and subjected to direct chemical coupling in vitro to form bispecific antibodies. The bispecific antibody thus formed was able to bind ErbB2 receptor overexpressing cells and normal human T cells, and was able to trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques have also been described for the production and isolation of bivalent antibody fragments directly from recombinant cell culture. For example, divalent heterodimers have been generated using the leucine zipper structure. Kostelny et al., J. Immunol., 148 (5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. The "bifunctional antibody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90 :6444-6448 (1993) provides an alternative mechanism for making bispecific/bivalent antibody fragments. The fragment comprises a heavy chain variable region ( _VH ) connected to a light chain variable region ( _VL ) by a linker so short as to not allow pairing between the two regions of the same chain. Thus, the _VH and _VL regions of one fragment have to pair with the complementary _VL and _VH regions of the other fragment, thereby forming two antigen-binding sites. Another strategy to make bispecific/bivalent antibody fragments using single-chain Fv (sFv) dimers has been reported. See Gruber et al., J. Immunol., 152 :5368 (1994).

Antibodies with more than two valencies are also within the scope of the invention. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147 :60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes on a given molecule. Alternatively, an anti-protein arm can be combined with an arm that binds a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or an Fc receptor for IgG (FcγR ), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus the cellular defense mechanism on cells expressing specific proteins. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing specific proteins. Such antibodies possess a protein binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds the protein of interest and further binds tissue factor (TF).

6) Heteroconjugate antibody

Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies. For example, one antibody in a heteroconjugate can be conjugated to avidin and the other to biotin. For example, such antibodies have been planned to target immune system cells to unwanted cells, US Patent 4,676,980, and to treat HIV infection. WO 91/00360, WO 92/200373 and EP 0308936. It is contemplated that the antibodies may also be prepared in vitro using known methods of synthetic protein chemistry, including those utilizing cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in US Patent No. 4,676,980. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in US Patent No. 4,676,980 along with a number of crosslinking techniques.

(7) Function modification of effector

It is desirable to modify the antibodies of the invention according to effector function in order to enhance the effectiveness of the antibodies in the treatment of cancer. For example, cysteine residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibodies thus produced have increased internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med,. 176 :1191-1195 (1992) and Shopes, J. Immunol. 148 :2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53 :2560-2565 (1993). Alternatively, antibodies can be designed with dual Fc regions such that the antibody has enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3 :219-230 (1989).

(8) Immunoconjugates

The invention also relates to immunoconjugates comprising antibodies conjugated to cytotoxic agents, such as chemotherapeutic agents, toxins (e.g., bacterial, fungal, plant or animal enzymatically active toxins, or their fragments) or radioactive isotopes (ie, radioconjugates).

Chemotherapeutic agents useful for generating such immunoconjugates include BCNU, streptozoicin, vincristine, vinblastine, doxorubicin, and 5-fluorouracil.

Enzyme-active toxins and their fragments that can be used include: diphtheria toxin A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain , lotus root toxin A chain, α-toxin, tung tree (Aleutites fordii) protein, caryophyllin protein, American pokeweed (Phytolaca Americana) protein (PAPI, PAPII, PAP-S), bitter melon (momordica charantia) inhibition Factor, jatrophin, crotonin, sapaonaria officinalis inhibitors, gelonin, mitogellin, limiterin, phenomycin, enomycin, and trichothecenes Tricothecenes.

The conjugates of antibodies and cytotoxic agents can be linked by various bifunctional protein coupling agents, such as: N-succinimidyl-3-(2-pyridyldimercapto)propane ester (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidate (such as dimethyl iminoadipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde (glutaraldehyde)), bis-azides (such as bis (p-azidobenzoyl)hexamethylenediamine), bis-diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as tolylene 2 , 6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described by Vitetta et al., Science 238:1098 (1987). C ¹⁴ -labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetate (MX-DTPA) is one of the coupling agents for conjugating radionucleotides to antibodies. See WO94/11026. This linker may be a "cleavable linker" that facilitates intracellular release of cytotoxic drugs. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992)).

In addition, small molecule toxins such as calicheamicin, maytansine (US Patent 5,208,020), trichothene, and CC1065 are also expected to be used in the formulations of the present invention as conjugable toxins. In one embodiment, a full-length antibody or antigen-binding fragment thereof may be conjugated to one or more maytansinoid molecules (e.g., about 1-10 maytansinoid molecules per antibody molecule) molecular binding). Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansinoids, including maytansine, maytansinal, and derivatives and analogs thereof, isolated from natural sources, or prepared synthetically, have been described, see U.S. Patent No. 5,208,020 and references cited therein (see § 2 column 53 to column 3, line 10) and US Patents 3,896,111 and 4,151,042. Methods for preparing antibody-maytansinoid conjugates are also described in US Patent No. 5,208,020. In a preferred embodiment, the maytansinoid is attached to the antibody via a disulfide or other sulfur-containing linker group. For example, maytansinoids can be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with modified antibodies to generate maytansinoid-antibody immunoconjugates. Chari et al., Cancer Res. 52 : 127-131 (1992). Antibodies can be modified by known methods, antibodies containing free or protected thiol groups are then reacted with disulfide containing maytansinoids to generate the conjugates. The cytotoxicity of the antibody-maytansinoid conjugate can be measured in vitro or in vivo by known methods and can be determined by _IC50 .

Calicheamicin is another immunoconjugate of interest. Antibiotics of the calicheamicin family produce double-stranded DNA breaks at sub-picomolar concentrations. Structural analogs of calicheamicin that may be used include, but are not limited to, γ ₁ ¹ , α ₂ ¹ , α ₃ ¹ , N-acetyl-γ ₁ ¹ , PSAG, and θ ₁ ¹ (Hinman et al., Cancer Res. 53 : 3336-3342 (1993) and Lode et al., Cancer Res. 58 :2925-2928 (1998)). Other antineoplastic drugs that can be linked to antibodies include QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and cannot easily cross the plasma membrane. Thus, cellular uptake of these agents through antibody-mediated internalization greatly enhances their cytotoxic effects.

Immunoconjugates formed between antibodies and compounds having nucleolytic activity (eg, ribonucleases or DNA endonucleases, eg, deoxyribonuclease, DNase) are also contemplated.

Antibodies can also bind to highly radioactive atoms. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include At ²¹¹ , Bi ²¹² , I ¹³¹ , In ¹³¹ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , P ³² and Pb ²¹² and radioactive isotopes of Lu. When the conjugate is for diagnostics, it may contain a radioactive atom for scintigraphic studies, such as Tc ⁹⁹ or I ¹²³ , or for nuclear magnetic resonance (nmr) imaging (also called magnetic resonance imaging, mri) spin labels such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Radiolabels or other labels can be incorporated into the conjugates by known methods. For example, the peptides can be biosynthesized or chemically synthesized by chemical amino acid synthesis using suitable amino acid precursors comprising, for example, fluorine-19 or hydrogen in appropriate positions. Tc ⁹⁹ or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ labels can be attached via cysteine residues in the peptide. Yttrium-90 can be attached via lysine residues. The IODOGEN(R) method can be used to incorporate iodine-123, Fraker et al., Biohem. Biophys. Res. Commun. 80 :49-57 (1978). Other methods of conjugating radionuclides are described in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be produced by recombinant techniques or peptide synthesis. The length of DNA may comprise separate regions encoding the two parts of the conjugate, which regions may be adjacent to each other, or separated by a region encoding a linker peptide which does not disrupt the conjugate. desired properties of the compound.

In another embodiment, antibodies can be conjugated to "receptors" such as streptavidin for use in pretargeting of tumors, where the antibody-receptor conjugate is administered to the patient and then cleared with The agent removes unbound conjugate from circulation prior to administration of a "ligand" (eg, avidin) that binds to the cytotoxic agent (eg, radionucleotide).

(9) Immunoliposome

Antibodies disclosed herein can also be formulated as immunoliposomes. "Liposomes" are small vesicles composed of various types of lipids, phospholipids and/or surfactants, useful for delivering drugs to mammals. The components of liposomes are usually arranged in a bilayer formation, similar to the lipid distribution of biological membranes.

Antibody-containing liposomes are prepared by methods known in the art, for example Epstein et al., Proc. Natl. Acad. Sci. USA, 82 : 3688 (1985); 4030 (1980); and the methods described in US Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in US Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Extrusion of liposomes through a filter material of defined pore size produces liposomes with the desired diameter. Fab' fragments of antibodies of the invention can be attached to liposomes by a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257 :286-288 (1982). Chemotherapeutic agents such as doxorubicin are optionally contained within the liposomes. See Gabizon et al., J. National Cancer Inst. 81 (19):1484 (1989).

(10) Other antibody modifications

Other modifications to the antibodies herein are contemplated. For example, antibodies can be linked to various nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. Antibodies can also be encapsulated in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, which are passed through, For example, coacervation techniques or interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules, and poly-(methyl methacrylate) microcapsules, respectively). Such techniques and suitable formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of Pharmacy and Science 2000).

C. Freeze-dried preparation

The formulations described herein can also be prepared as reconstituted lyophilized formulations. The proteins or antibodies described herein are lyophilized and then reconstituted to produce the reduced viscosity stable liquid formulations of the invention. In this particular embodiment, a "pre-lyophilized formulation" is produced after the protein of interest has been prepared as described above. The amount of protein present in the pre-lyophilized formulation is determined taking into account the desired dosage, mode of administration, and the like. For example, the starting concentration of intact antibody may be from about 2 mg/ml to about 50 mg/ml, preferably from about 5 mg/ml to about 40 mg/ml and most preferably about 20-30 mg/ml.

(1) Preparation of freeze-dried preparation

The protein to be formulated is generally in solution. For example, in the increased ionic strength lowered viscosity formulations of the present invention, the protein may be present in a pH buffer solution having a pH of about 4-8, preferably about 5-7. Depending on, for example, the buffer and the desired tonicity of the formulation (eg, the tonicity of the reconstituted formulation), the buffer concentration can be from about 1 mM to about 20 mM, alternatively from about 3 mM to about 15 mM. Exemplary buffers and/or salts are those that are pharmaceutically acceptable and may be derived from suitable acids, bases and their salts, such as those defined by "pharmaceutically acceptable", acids, base or buffer.

In one embodiment, a lyoprotectant is added to the prelyophilized formulation. The amount of dissolved protectant in the prelyophilized formulation is generally such that upon reconstitution the resulting formulation is isotonic. However, hypertonic reconstituted formulations may also be suitable. Furthermore, the amount of lysoprotectant should not be so low that an unacceptable amount of protein degradation/aggregation occurs upon lyophilization. However, exemplary lyoprotectant concentrations in pre-lyophilized formulations are from about 10 mM to about 400 mM, alternatively from about 30 mM to about 300 mM, alternatively from about 50 mM to about 100 mM. Exemplary lysoprotectants include sugars and sugar alcohols such as sucrose, mannose, trehalose, glucose, sorbitol, mannitol. However, certain dissolution protectants can also increase the viscosity of the formulation under certain circumstances. Likewise, care should be taken in selecting a specific lysoprotectant that minimizes and neutralizes these effects. Additional lysoprotectants, also referred to herein as "pharmaceutically acceptable sugars", are described above under the definition of "lysoprotectant".

The ratio of protein to lysoprotectant can vary for each particular protein or antibody and lysoprotectant combination. The molar ratio of lysoprotectant to antibody can be from about 100 to About 1500 moles of lysoprotectant to 1 mole of antibody, and preferably from about 200 to about 1000 moles of lysoprotectant to 1 mole of antibody, eg, from about 200 to about 600 moles of lysoprotectant to 1 mole of antibody.

In a preferred embodiment, it is advisable to add a surfactant to a pre-lyophilized formulation. Alternatively, or in addition, surfactants may be added to the lyophilized and/or reconstituted formulations. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g. polysorbate 20 or 80); polyoxamers (e.g. poloxamer 188); Triton; sodium octyl glycoside; Tetraalkyl-, linoleyl-, or stearoyl-sulfobetaine; lauryl-, tetradecyl-, linoleyl-, or stearoyl-sarcosine ; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (eg laurylamidopropyl); myristamidopropyl-, palmidopropyl -, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-taurate or disodium methyloleyl-taurate; and MONAQUA ^TM series (Mona Industries, Inc., Paterson, New Jersey), polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (eg, Pluronics, PF68, etc.). The amount of surfactant added is such that it reduces particulate formation of the reconstituted protein and minimizes particulate formation after reconstitution. For example, the amount of surfactant present in the prelyophilized formulation is about 0.001-0.5%, alternatively about 0.005-0.05%.

A mixture of a lysis protectant (such as sucrose or trehalose) and a bulking agent (such as mannitol or glycine) may be used in the preparation of the prelyophilized formulation. The bulking agent may allow for the creation of a uniform lyophilized mass without excessive voids in it. Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Phannaceutical Sciences 16th edition, Osol, A.Ed. (1980), may be included in the prelyophilized formulation (and /or lyophilized formulations and/or reconstituted formulations), provided they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers that are nontoxic to recipients at the dosages and concentrations employed include: additional buffers; preservatives; auxiliary solvents; antioxidants, including ascorbic acid and methionine ; chelating agents such as EDTA; metal complexes such as Zn-protein complexes; biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium.

Formulations herein may also contain more than one protein, preferably those having complementary activities that do not adversely affect another protein, as necessary for the particular condition being treated. For example, it may be desirable to provide two or more antibodies that bind to a desired target (eg, receptor or antigen) in a single formulation. Such proteins are suitably present in the composition in amounts effective for the intended purpose.

Preparations for in vivo administration must be sterile. This is easily accomplished by filtration with a sterile filter membrane, either before lyophilization and reconstitution, or afterward. Alternatively, for example, the complete mixture can be sterilized by autoclaving at about 120[deg.] C. for 30 minutes with the exception of the protein.

After the protein, optional lysoprotectant, and other optional ingredients are mixed together, the formulation is lyophilized. For this purpose, many different freeze-dryers are available, eg Hull50 ^™ (Hull, USA) or GT20 ^™ (Leybold-Heraeus, Germany) freeze-dryers. Lyophilization is achieved by freezing the formulation followed by sublimation of ice from the frozen contents at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic or collapse temperature of the formulation. Typically, shelf temperatures for primary drying range from about -30°C to 25°C (provided the product is kept frozen during primary drying), under appropriate pressures, typically ranging from about 50 to 250 mTorr. The formulation, size and type of container holding the sample (eg, glass vial), and volume of liquid will largely determine the time required for drying, which can range from hours to days (eg, 40-60 hours). Optionally, depending on the desired residual moisture level in the product, a second drying may also be performed. The temperature at which the second drying is carried out ranges from about 0 to 40°C, depending mainly on the type and size of the container and the type of protein used. For example, the storage temperature may be about 15-30°C (eg, about 20°C) throughout the water removal stage of lyophilization. The time and pressure required for the second drying is such that a suitable freeze-dried mass will be produced, depending on eg temperature and other parameters. The second drying time is determined by the desired residual moisture level in the product and generally requires at least about 5 hours (eg, 10-15 hours). The pressure may be the same as used during the primary drying step. Depending on the formulation and vial size, lyophilization conditions can vary.

2. Reconstitution of lyophilized formulations

Prior to administration to a patient, the lyophilized formulation is reconstituted with a pharmaceutically acceptable diluent such that the protein concentration in the reconstituted formulation is at least about 50 mg/ml, such as from about 50 mg/ml to about 400 mg/ml, as Select from about 80 mg/ml to about 300 mg/ml, alternatively from about 90 mg/ml to about 150 mg/ml. Such high protein concentrations in reconstituted formulations are believed to be particularly useful when it is desired to deliver the reconstituted formulation subcutaneously. However, for other routes of administration, such as intravenous administration, lower protein concentrations in the reconstituted formulation may be desirable (e.g., about 5-50 mg/ml of protein in the reconstituted formulation, or about 10-40 mg /ml). In certain embodiments, the protein concentration in the reconstituted formulation is significantly higher than in the pre-lyophilized formulation. For example, the protein concentration in the reconstituted formulation may be about 2-40 times, alternatively 3-10 times, alternatively 3-6 times (e.g., at least three times or at least four times) the protein concentration in the pre-lyophilized formulation. ).

Reconstitution generally occurs at about 25°C to ensure complete hydration, although other temperatures are free to use. The time required for reconstitution depends, for example, on the type of diluent, amount of excipients and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above, aromatic alcohols such as benzyl alcohol or phenolic alcohols are preferred preservatives. The amount of preservative used was determined by evaluating the compatibility of different preservative concentrations with the protein and preservative efficiency tests. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it may be present in an amount of about 0.1-2.0%, preferably about 0.5-1.5%, but most preferably about 1.0-1.2%.

Preferably, the reconstituted formulation is less than 6000 particles per vial with a particle size > 10 μm.

D. Liquid formulations

Therapeutic formulations for preservation are prepared by mixing the active ingredient with the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 18th edition, Mack Publishing Co., Easton, Pa. 18042 [1990]). Acceptable carriers, excipients or stabilizers that are nontoxic to recipients at the dosages and concentrations employed include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives , isotonizing agents, stabilizers, metal complexes (eg Zn protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.

When the therapeutic agent is an antibody fragment, a minimal inhibitory fragment that specifically binds to the binding region of the protein of interest is preferred. For example, according to the variable region sequence of an antibody, antibody fragments or even peptide molecules can be designed to retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and/or produced by recombinant DNA techniques (see, eg, Marasco et al., Proc. Natl. Acad. Sci. USA 90 :7889-7893 [1993]).

Buffers are used to control the pH within a range that optimizes efficacy, especially if stability is pH dependent. The preferred concentration range of the buffer is from about 50 mM to about 250 mM. Suitable buffering agents for use in the present invention include organic and inorganic acids, and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Alternatively, the buffer may consist of histidine and trimethylamine salts, such as Tris.

Preservatives are added to inhibit microbial growth, generally in the range of 0.2%-1.0% (w/v). Suitable preservatives for use in the present invention include, for example, octadecyldimethylphenylammonium chloride; hexacene quaternary ammonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), chloride Benzethonium; Thimerosal, Phenol, Butyl Methanol, or Benzyl Alcohol; Hydrocarbylparabens, such as Methylparaben or Propylparaben; Catechol; Resorcinol; Cyclohexanol , 3-pentanol, and m-cresol. .

A tonicity agent, sometimes called a "stabilizer," is included to adjust or maintain the liquid tonicity of the composition. When working with large, charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because of their ability to interact with charged groups on the side chains of amino acids, thereby reducing the potential for intra- and intermolecular interactions. The tonicity agent may be present in any amount between 0.1% and 25%, preferably between 1 and 5% by weight, taking into account the relative amounts of the other ingredients. Tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional excipients include agents that may act as one or more of (1) fillers, (2) dissolution enhancers, (3) stabilizers, and (4) agents that inhibit denaturation or attachment to the container walls. Stabilizers can range from 0.1 to 10,000 parts by weight of active protein or antibody. Typical stabilizers include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine , leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose , xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclopolyols (e.g., cyclohexanehexanol), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione Peptides, lipoic acid, sodium thioacetate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic Polymers, such as polyvinylpyrrolidone; monosaccharides (for example, xylose, mannose, fructose, glucose; disaccharides (for example, lactose, maltose, sucrose); trisaccharides, such as raffinose; Refined or dextran.

Contains non-ionic surfactants or detergents (also known as "wetting agents") to aid dissolution of the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also allows the formulation to be subjected to shear surface stress without resulting in active therapeutic Denaturation of proteins or antibodies. The nonionic surfactant ranges from about 0.05 mg/ml to about 1.0 mg/ml, preferably from about 0.07 mg/ml to about 0.2 mg/ml.

Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), Pluronic® polyls, Triton®, polyoxyethylene sorbitan mono Ether (polyoxyethylene sorbitan monoether) (Tween-20, Tween-80, etc.), lauromacrogol 400, polyoxyl 40 stearate (ester), polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil) 10, 50 and 60 , glyceryl monostearate, fatty acid sugar esters, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for formulations to be used for in vivo administration, they must be sterile. The formulation may be sterilized by filtration through a sterile filter membrane. The therapeutic compositions herein are generally presented in containers having sterile access ports, eg, bags for intravenous solutions or vials with stoppers which can be pierced by a hypodermic needle.

The route of administration is according to known and accepted methods, for example by single or multiple bolus administration (bolus), or infusion in a suitable manner over a long period of time, for example, by subcutaneous, intravenous, intraperitoneal Intramuscular, intraarterial, intralesional or intraarticular routes, topically, by inhalation or by sustained or delayed release methods.

The formulations herein may also contain more than one active compound, preferably those active compounds with complementary activities that do not adversely affect each other, as necessary for the particular condition to be treated. Alternatively, or in addition, the composition may include a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in the composition in amounts effective for the intended purpose.

The active ingredient may also be encapsulated in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions through, For example, coacervation techniques or interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules, and poly-(methyl methacrylate) microcapsules, respectively). This technique is disclosed in Remington's Pharmaceutical Sciences 18th edition, supra.

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers comprising the antibody in the form of shaped objects such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-glucose Amino acid and γ-ethyl-L-glutamic acid copolymer, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, such as LUPRON DEPOT ^TM (made of lactic acid-glycolic acid copolymer and acetic acid Injectable microspheres composed of leuprolide), and poly-D-(-)-3-hydroxybutyrate. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rpg 120. Johnson et al., Nat.Med. 2 : 795-799 (1996); Yasuda et al., Biomed.Ther. 27 : 1221-1223 (1993); Hora et al., Bio/Technology 8 : 755-758 (1990); Cleland, " Design and Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems, "in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds., (Plenum Press: New York, 1995), pp.439-462; WO 97/03692; WO 96/40072; WO 96/07399; and US Patent No. 5,654,010.

Sustained-release formulations of these proteins can be developed using polylactic-coglycolic acid (PLGA) polymers due to the biocompatibility and broad biodegradability of PLGA. The degradation products of PLGA, lactic acid and glycolic acid, are quickly cleared from the body. Furthermore, depending on its molecular weight and composition, the degradability of this polymer can be tuned from months to years. Lewis, "Controlled release of bioactive agents fromlactide/glycolide polymer", in Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker; New York, 1990), M. Chasin and R. Langer (Eds.) pp. 1-41.

Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, and certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies remain in the body for long periods of time, they may denature or aggregate as a result of exposure to humidity at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanisms involved, rational strategies can be devised for stability. For example, if the mechanism of aggregation is found to be intermolecular S-S bond formation via thio-disulfide interchange, it may be possible to modify sulfhydryl residues, lyophilize from acidic solutions, control water content, utilize appropriate additives, and develop specific aggregates. material matrix composition to achieve stabilization.

Liposome or proteinoid compositions can also be used to formulate the proteins or antibodies disclosed herein. See US Patent Nos. 4,925,673 and 5,013,556.

The stability of the proteins and antibodies described here can be enhanced through the use of non-toxic "water-soluble polyvalent metal salts". Examples include Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , Cu ²⁺ , Sn ²⁺ , Sn ⁴⁺ , Al ²⁺ , and Al ³⁺ . Examples of anions that can form water-soluble salts with the above-mentioned polyvalent metal cations include those formed from inorganic acids and/or organic acids. Such water soluble salts have a solubility in water (20°C) of at least about 20 mg/ml, optionally at least about 100 mg/ml, optionally at least about 200 mg/ml.

Suitable inorganic acids that can be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid, and phosphoric acid. Suitable organic acids that can be used include aliphatic carboxylic acids and aromatic acids. Aliphatic acids under this definition can be defined as saturated or unsaturated _C2-9 carboxylic acids (eg, aliphatic mono-, di- and tricarboxylic acids). For example, exemplary monocarboxylic acids under this definition include saturated _C2-9 monocarboxylic acids, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, capryonic acid, and not Saturated C _2-9 monocarboxylic acids, acrylic acid, propriolic methacrylic acid, crotonic acid and isocrotonic acid. Exemplary dicarboxylic acids include saturated _C2-9 dicarboxylic acids, malonic acid, succinic acid, glutaric acid, adipic acid, and pimelic acid, while unsaturated _C2-9 dicarboxylic acids include maleic acid dioic acid, fumaric acid, citraconic acid and methyl fumaric acid. Exemplary tricarboxylic acids include saturated C _2-9 tricarboxylic acids, propanetricarboxylic acid and 1,2,3-butanetricarboxylic acid. In addition, carboxylic acids of this definition also contain one or two hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, hydroxyglutaric acid, malic acid, tartaric acid, and citric acid. Aromatic acids under this definition include benzoic and salicylic acids.

Commonly used water-soluble polyvalent metal salts that can be used to help stabilize the encapsulated polypeptides of the present invention include, for example: (1) metal salts of halogenated inorganic acids (e.g., zinc chloride, calcium chloride), sulfates, Nitrates, phosphates, and thiocyanates; (2) metal salts of aliphatic carboxylic acids (for example, calcium acetate, zinc acetate, calcium proprionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate); and (3) Metal salts of aromatic carboxylic acids, benzoates (eg, zinc benzoate) and salicylates.

E. Medical methods

For the prophylaxis or treatment of disease, the appropriate dosage of the active agent will depend on the type of disease to be treated as defined above, the severity and course of the disease, whether the agent is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and Response to medication, and judgment of the attending physician. The agent is suitably administered to the patient at one time or over the course of a therapeutic series.

A preferred method of treatment is the treatment of IgE-mediated diseases. IgE-mediated diseases include atopic disorders, which are characterized by an inherited predisposition to mount immune responses to many commonly occurring naturally occurring inhaled and ingested antigens, and to persistently produce IgE antibodies. Specific atopic diseases include allergic asthma, allergic rhinitis, atopic dermatitis, and allergic gastroenteropathy. Atopic patients often have polysensitivity, meaning they have IgE antibodies, and symptoms resulting therefrom, to many environmental allergens, including pollens, fungi (eg, molds), animal and insect debris, and certain foods.

However, diseases associated with increased IgE levels are not limited to those with a genetic (atopic) etiology. Other diseases associated with increased IgE levels that appear to be IgE-mediated and that can be treated with the formulations of the invention include hypersensitivity reactions (e.g., anaphylactic hypersensitivity reactions), eczema, urticaria, allergic bronchial Allergic bronchopulmonary aspergillosis, parasitic diseases, interstitial cystitis, hyper-IgE syndrome, ataxia-telangiectasia, Wiskott-Aldrich syndrome, thymic lymphoid Thymic alymphoplasia, IgE myeloma, and graft-versus-host reaction.

Allergic rhinitis, also known as allergic rhinoconjunctivitis or hay fever, is the most common form of atopic reaction to inhaled allergens, and its severity and duration often correspond to the intensity and duration of exposure to the allergen related. It is a chronic condition that can first appear at any age, but usually begins in childhood or adolescence. Typical episodes include profuse watery rhinorrhea, paroxysmal sneezing, nasal congestion, and nasal and palate exhaustion. Mucus drainage behind the nose also causes strep throat, throat clearing, and coughing. There may also be symptoms of allergic blepharoconjunctivitis, with intense itching of the conjunctiva and eyelids, redness, tearing, and photophobia. Severe attacks are often accompanied by general malaise, weakness, fatigue, and sometimes muscle pain during severe sneezing.

Asthma, also known as reversible obstructive airway disease, is characterized by hyperresponsiveness of the tracheobronchial tree to respiratory irritants and bronchoconstricting chemicals, producing episodes of wheezing, dyspnea, chest tightness, and coughing that are naturally reversible , or reversible by medical treatment. It is a chronic condition involving the entire airway, ranging in severity from occasional mild transient reactions to severe, chronic, life-threatening bronchial obstruction. Asthma and atopy can coexist, but only about half of patients with asthma are also atopy, and an even smaller proportion of patients with atopy also have asthma. However, atopy and asthma are not completely independent, as asthma occurs more frequently in atopic individuals than in non-atopic individuals, especially during childhood. Asthma has historically been further broken down into two subgroups, extrinsic and intrinsic.

Extrinsic asthma, also known as allergic, atopic, or autoimmune asthma, is a description for patients who typically develop asthma at a young age, usually in early childhood or childhood. Other manifestations of atopy, including eczema or allergic rhinitis, often occur concurrently. Asthma attacks can occur during pollen season, in the presence of animals, or when exposed to house dust, feather pillows, or other allergens. Skin testing revealed a positive rash and erythematous reaction to the causative allergen. Interestingly, total serum IgE concentrations are often increased but sometimes normal.

Intrinsic asthma, also known as nonallergic or idiopathic asthma, typically first occurs in adulthood, followed by overt respiratory infections. Symptoms include chronic or recurrent bronchial obstruction unrelated to pollen season or exposure to other allergens. Skin tests for common atopic allergens were negative, and serum IgE concentrations were normal. Additional symptoms include bloody sputum and eosinophilia. Other schemes for classifying asthma into subgroups, like aspirin-sensitive, exercise-induced, infectious, and merely psychological, define extrinsic triggers that affect some patients more than others factor.

Finally, it is important to note that while some classifications previously only linked allergic asthma to IgE dependence, there are now robust and statistically significant data showing that IgE and asthma (allergic and nonallergic ) between the correlation. Chapter 27, "The Atopic Diseases", A.I. Terr in Medical Immunology, 9th Ed., Simon and Schuster, Stites et al., Ed. (1997). Thus, the term "IgE-mediated disease" for the purposes of this application includes allergic and non-allergic asthma.

Signs of an asthma attack include shortness of breath, audible wheezing, and use of accessory respiratory muscles. A rapid pulse and elevated blood pressure are also generally present, as are elevated levels of peripheral blood eosinophils and increased nasal discharge. Lung function showed a decrease in lung flux and forced expiratory volume in 1 second. Total vital capacity and functional residual capacity are generally normal or slightly increased, but may decrease in extreme bronchospasm.

The pathology of asthma can be differentiated into early phase and late phase responses. The early phase is characterized by smooth muscle contraction, edema, and hypersecretion, while the late response is characterized by cellular inflammation. Asthma can be induced by a variety of nonspecific triggers, including infections (eg, viral respiratory infections), physiological factors (eg, exercise, hyperventilation, deep breathing, psychological factors), atmospheric factors (eg, sulfur dioxide, ammonia, cold air, ozone, distilled water vapor), ingestion (eg, propranolol, aspirin, NSAIDs), experimental inhalation (eg, hypertonic solutions, citric acid, histamine, methacholine, prostaglandin F _2α ) and occupational inhalants (eg, isocyantes). Various other occupational or environmental allergens that cause allergic asthma include, animal products, insect dust, marine life, plant products, fruits, seeds, leaves and pollen, organic dyes and inks, microbial agents, enzymes, therapeutics, Bactericides, inorganic and organic chemicals.

Atopic dermatitis, also known as eczema, neurodermatitis, atopic eczema, or Besnier's prurigo, is a common skin disorder that is specific to patient populations with familial and atopic immunological features. The essential feature is a pruritic cutaneous inflammatory response that induces a characteristic symmetrically distributed rash that favors certain sites. Excessive production of IgE by B lymphocytes is also common. Although atopic dermatitis is classified as an atopic cutaneous manifestation due to its association with allergic rhinitis and asthma and high IgE levels, however, the severity of dermatitis does not necessarily correlate with skin test exposure to allergens, desensitization Therapy cannot be effectively treated (unlike other allergic diseases). Although high serum IgE levels are confirmatory for the diagnosis of allergic asthma, normal levels do not rule it out. Onset of the disease can occur at any age and the lesions begin acutely with erythematous edematous papules or scaly plaques. Itching leads to exudate and crusting, which then leads to chronic lichenoid sclerosis. At the cellular level, acute lesions are edematous, with dermis infiltrated by monocytes, CD4 lymphocytes. Neutrophils, eosinophils, plasma cells, and basophils are rare, but degranulated mast cells are present. Chronic lesions are characterized by epidermal hyperplasia, cutaneous hyperkeratosis, and parakeratosis, with dermis infiltrated by monocytes, Langerhans cells, and mast cells. There may also be fibrosis in focal areas, including involving the perineurium of small nerves.

Allergic gastroenteropathy, also known as eosinophilic gastroenteropathy, is an uncommon form of atopy in which multiple IgE food sensitivities are associated with localized gastrointestinal mucosal reactions. It is rare in adults, but is more common, but transient, in infants. This condition arises when ingested food allergens react with local IgE antibodies on the jejunal mucosa to release mast cell regulators, resulting in digestive symptoms shortly after a meal. Chronic inflammation from continued exposure, leading to loss of gastrointestinal protein and hypoproteinaceous edema. There can be sufficiently significant blood loss through the inflamed intestinal mucosa to cause iron deficiency anemia. Hypersensitivity reactions occur locally in the upper gastrointestinal mucosa after exposure to the allergen, but can be resolved by avoiding the allergen.

Anaphylaxis and urticaria are clearly IgE-mediated but are not genetically determined and there is no preference for atopic individuals. Anaphylaxis is an acute systemic allergic reaction involving several organ systems, usually the cardiovascular system, respiratory system, skin system, and gastrointestinal system. The response is immune-mediated and occurs upon exposure to an allergen to which the patient has previously been sensitized. Urticaria and angioedema are physical swelling, erythema, and itching caused by histamine-stimulated receptors on superficial blood vessels and are hallmark skin features of systemic allergies. Anaphylaxis is an IgE-mediated reaction to drugs, insect venoms, or foods that occurs simultaneously in multiple organs. It is suddenly caused by allergen-induced IgE loading on mast cells, leading to profound and life-threatening changes in the function of various vital organs. Vascular collapse, acute airway obstruction, cutaneous vasodilation and edema, and gastrointestinal and genitourinary muscle spasms occur almost simultaneously, although not necessarily to the same extent.

Symptoms of anaphylaxis include angioedema and lung hyperinflation with mucus plugs in the airways and partial atelectasis. At the cellular level, the lungs behave similarly to during an acute asthma attack, with bronchial submucosal gland hypersecretion, mucosal and submucosal edema, peribronchial vascular congestion, and eosinophilia in the bronchial wall. Pulmonary edema and hemorrhage may be present. There may also be bronchial muscle spasm, hyperinflation, or even alveolar rupture. Important features of anaphylaxis in humans include edema, vascular congestion, and eosinophilia in the lamina propria of the larynx, trachea, epiglottis, and tongue.

Exposure to an allergen can be through ingestion, injection, inhalation, or contact with the skin or mucous membranes. The reaction begins within seconds or minutes of exposure to the allergen. There may be an initial fear or feeling of near-death followed quickly by symptoms in one or more target organ systems: cardiovascular, respiratory, cutaneous, and gastrointestinal.

The allergens that cause allergic reactions are different from those commonly associated with atopy. Food, drugs, insect venom or latex are common sources. Food allergens include those found in crustaceans, molluscs (e.g., lobster, shrimp, crab), fish, legumes (e.g., peanuts, peas, broad beans, licorice), seeds (e.g., sesame, cottonseed, caraway seeds, pions, flaxseeds, sunflower seeds), nuts, berries, egg whites, buckwheat and milk. Drug allergens include those present in heterologous proteins and polypeptides, polysaccharides and hapten drugs. Insect allergens include insects of the order Hymenoptera, including bees, wasps, hornets, wasps, and fire ants.

Epinephrine is the typical treatment for anaphylaxis, and antihistamines or other histamine blockers are generally prescribed for less severe urticaria or angioedema reactions.

F. Combination therapy

The methods of the present invention can be combined with known methods of treating IgE-mediated diseases, either as a combined or additional therapeutic step, or as an additional component of a therapeutic formulation.

For example, antihistamines, particularly non-sedating antihistamines, may be administered prior to, or in proportion to, the anti-IgE antibodies of the invention. Suitable antihistamines include those belonging to the class of alkylamines (eg, chlorphenamine), ethanolamines (eg, diphenhydramine), and phenothiazines (eg, promethazine). Many antihistamines antagonize the pharmacological effects of histamine by blocking histamine receptor sites on effector cells, and other common antihistamines work by blocking the release of histamine from mast cells, the Mast cells have been sensitized or armed with allergen-specific IgE (eg, cromolyn). Examples of antihistamines include astemizole, piperheptidine maleate, bropheniramine maleate, chlorpheniramine maleate, cetirizine hydrochloride, clemastine fumarate, dipheniramine hydrochloride, Dexbrompheniramine maleate, dexchlorpheniramine maleate, chlorphylline diphenhydramine, diphenhydramine hydrochloride, duxilamine succinate, fexofendadine hydrochloride, terphenadine hydrochloride, hydroxyzine hydrochloride , loratidine, minkejing hydrochloride, tripyrolidine citrate, tripyrolidine hydrochloride, propyrrolidine hydrochloride.

Specific symptoms of IgE-mediated disease (eg, early phase reactions) can be improved with sympathomimetic or bronchodilatory drugs. Epinephrine is a broad-acting alpha and beta adrenergic drug that is usually administered subcutaneously in 0.2 to 0.5 mL of a 1:100 aqueous solution. Long-acting forms of epinephrine (eg, butaline) may also be used as a 1:200 suspension when a longer-lasting effect is desired. Suitable other beta-adrenergic drugs include nasal administration (eg, hand-held nebulizer, intermittent positive-pressure breathing device, or metered-dose inhaler) or oral albuterol, pirbuterol, metaproterenol, salmeterol , neoisoproterenol and formoterol.

Bronchodilation can also be achieved by the administration of xanthines, especially by co-administration with the sympathomimetic drugs described above. Examples of xanthines include aminophylline (intravenous injection, 250-500 mg) and theophylline (oral, 10-20 μg/ml serum concentration).

Other symptoms from various IgE-mediated diseases (eg, late phase reactions) can be attenuated by treatment with glucocorticoids or other drugs with anti-inflammatory effects. Prednisone (30-60 mg per day) is administered systemically for severe episodes, while for long-term maintenance therapy clodimil dipropionate, fluroxycortisol acetonate, and flunidine are administered in aerosol form Shrinkage. Other steroids with anti-inflammatory effects include: betamethasone, budesonide, dexamethasone, fludrocortisone acetate, flunisolide, fluticasone propionate, hydrocortisone, methylprednisolone, hydrocortisone Prednisone, prednisone, fludrocortisol.

Non-steroidal anti-inflammatory drugs that may also be used in combination with the methods of treatment of the present invention include acetaminophen, acetylsalicylic acid, bromfenac sodium, diclofenac sodium, diflunisal, etodolac, benzene Calcium oxyhydroatropate, flurbiprofen, ibuprofen, indole chloroformyl, ketoprofen, meclofenac sodium, mefenamic acid, nabumetone, naproxen, formazan Naproxen sodium, hydroxybutazone, phenylbutzone, piroxicam, sulindac, meluoyl pirinate sodium.

In addition, decongestants (eg, phenylephrine, phenylpropanolamine, pseudoephadrin), cough suppressants (eg, methorphan, codeine, or hydrocodone), or analgesics (eg, acetaminophen , aspirin) to maximize therapeutic benefit.

Allergen desensitization is a form of treatment in which an allergen is injected into a patient for the purpose of reducing or eliminating the allergic reaction. This is also called allergen immunotherapy, desensitization, or allergy shot therapy. It is often used in combination with other allergy treatments, but often not as a primary treatment. It has been used successfully in situations where allergen avoidance is not possible. Typical allergen desensitization therapy involves subcutaneous injections of sterile allergens once or twice weekly in increasing doses until a dose is sufficient to produce transient minimal local inflammation at the injection site. This dose is then given every 2-4 weeks on a maintenance schedule. Allergic desensitization is most commonly used in the treatment of allergic asthma and allergic rhinitis, although it has also been used successfully in the treatment of anaphylaxis. Desensitization is also effectively performed by the use of an adjuvant, such as Freund's incomplete adjuvant, which is an emulsion of aqueous antigen in mineral oil. Its physiological effect creates an insoluble liquid reservoir from which micro-droplets of the allergen can be gradually released. Another form of allergen desensitization is the polymerization of monomeric allergens with glutaraldehyde to produce molecules that are relatively low in allergenicity (eg, cause anaphylaxis), while maintaining effective levels of immunogenicity.

G. Drug dosage:

Dosages and desired drug concentrations of the pharmaceutical compositions of this invention may vary depending upon the particular use envisioned. Determination of an appropriate dosage or route of administration is well within the ability of the ordinary skilled artisan. Animal experiments provide reliable guidance for determining effective doses for human therapy. Can be carried out according to the principles presented in Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp.42-46 Interspecies conversion of effective dose.

When performing in vivo administration of the polypeptides or antibodies described herein, normal dosage numbers may range from about 10 ng/kg per day up to about 100 mg/kg of mammalian body weight or more, preferably about 1 mg/kg/day, depending on the route of administration. to 10mg/kg/day. Guidance on particular dosages and methods of administration have been provided in the literature, see, eg, US Patent Nos. 4,657,760; 5,206,344 or 5,225,212. It is within the scope of this invention that different formulations are effective for different treatments and different diseases, and that administration intended to treat a particular organ or tissue may have to be administered in a manner different from that used to treat another organ or tissue. medication. Furthermore, dosing may be by one or more separate administrations, or by continuous infusion. For repeated applications over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms is produced. However, other dosing regimens are also useful. The progress of the therapy can be easily monitored by conventional techniques and analyses.

H. Administration of the formulation

The formulations of the invention, including but not limited to reconstituted formulations, are administered according to known methods, e.g., as a bolus intravenously, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular , intrasynovial, intrathecal, oral, topical or inhalation, continuous infusion over a period of time, for administration to a mammal in need of treatment with the protein, preferably a human .

In a preferred embodiment, the formulation is administered to the mammal by subcutaneous administration (eg, under the skin). For this purpose, the formulation can be injected with a syringe. However, other devices for administering the formulation are available, such as infusion devices (e.g., Inject-ease ^™ and Genject ^™ devices); injection pens (e.g., GenPen ^™ ); automatic injection devices, needle-free devices (e.g., MediJector ^™ and BioJector ^™ ) and subcutaneous patch delivery systems.

In a particular embodiment, the invention relates to kits for single dosage administration units. Such kits include containers of aqueous formulations of therapeutic proteins or antibodies, including single or multi-lumen prefilled syringes. Exemplary prefilled syringes are available from Vetter GmbH, Ravensburg, Germany.

The appropriate dosage ("therapeutically effective amount") of the protein will depend, for example, on the condition being treated, the severity and course of the condition, whether the protein is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the protein. The response, the type of protein used, and the judgment of the attending physician. The protein can be administered to the patient at one time or over a series of medical procedures, or can be administered to the patient at any time since diagnosis. The protein can be administered as the sole treatment, or in combination with other drugs or treatments that are useful in treating the current condition.

Alternative proteins are antibodies, and about 0.1-20 mg/kg is an initial candidate dose for administration to a patient, eg, whether in one or multiple separate administrations. However, other dosing regimens are also useful. The progress of therapy can be easily monitored by conventional techniques.

Uses of anti-IgE agents (eg, rhuMAbE-25, rhMAbE-26, Hu-901 ) include the treatment or prevention of, eg, IgE-mediated allergic diseases, parasitic infections, interstitial cystitis and asthma. Depending on the disease or condition being treated, a therapeutically effective amount (eg, about 1-15 mg/kg) of anti-IgE antibody is administered to the patient.

I. Products

In another embodiment of the invention, an article of manufacture comprising said formulation is provided, preferably with instructions for its use. The article includes a container. Suitable containers include, for example, bottles, vials (eg, dual-chambered vials), syringes (eg, single- or dual-chambered syringes), and test tubes. The container can be made of various materials such as glass or plastic. The container containing the formulation and a label on, or associated with, the container may bear instructions for reconstitution and/or use. The label may further state that the formulation is useful or intended for subcutaneous administration. The container with the formulation can be a multiple-use vial that allows for repeated administrations (eg, 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (eg, BWFI). Upon mixing the diluent and lyophilized formulation, the final protein concentration in the reconstituted formulation will generally be at least 50 mg/ml. The article of manufacture may further comprise other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The present invention will be more fully understood by reference to the following examples. However, these examples should not be viewed as limiting the scope of the present invention. All content cited herein is hereby incorporated by reference in its entirety.

In another embodiment, the present invention provides an article of manufacture comprising a formulation described herein for administration in an autoinjector device. An auto-injector can be described as an injection device that, when activated, delivers its contents without additional necessary action by the patient or the administerer. They are particularly suitable for self-medication of therapeutic formulations when the rate of administration must be constant and over a relatively long period of time.

Example 1

Preparation of Anti-IgE rhuMAbE25 ("E25") Formulations

From E25 bulk residual Lot K9094A (40mg/ml rhuMAb E25, 85mM trehalose, 5mM histidine, pH6, 0.01% Tween 20) or rhuMAbE25Q-Pool (5mg/ml rhuMAb E25, 25mM Tris, 200mM NaCl) A preparation of the monoclonal anti-IgE antibody rhuMAbE25 was prepared.

Aqueous solutions of rhuMAbE25 were prepared by dialysis at 2-8°C into different buffers (20 mM His-HCl and 200 mM Arg-HCl, pH 6.0) using a Slide-A-Lyzer dialysis cassette (Pierce). Samples were then transferred to the sample reservoirs of Centricon-30 centrifugal microconcentrators (Amicon). The protein was concentrated by spinning a Centricon-3 concentrator at 4000-500Og until the desired protein concentration was reached.

The sample was then concentrated to ~150 mg/ml rhuMAb E25 using ultrafiltration. Tween 20 was added to each preparation to a final concentration of 0.02%. All formulations were filtered, aseptically filled into 3cc FormaVitrum vials in a Class 100 room, and stoppered with 13-mM Diakyo.

Example 2

Methods and materials:

Stability study: 1ml of all formulations were filled into 3ccFormaVitrum glass vials in a Class 100 aseptic filling room and stoppered with 13-mm Diakyo stoppers. Place vials in light-tight containers at -70°C, 2-8°C, 15°C, and 30°C.

Agitation Studies: Aliquots of each formulation were placed in glass vials. Vials were agitated horizontally at room temperature on a Glas-Col Bench Top shaker. Set the shaker to 70 with a 30 cm arm length (maximum). After agitation, samples were examined and analyzed according to the following protocol.

Freeze-thaw studies: Samples of E25 were subjected to three freeze-thaw cycles. Each cycle consisted of overnight freezing at -70°C, and subsequent thawing at room temperature for approximately one hour. After each cycle, the samples were visually inspected using a light box to assess the color and clarity of the liquid. Turbidity and soluble aggregates were measured following the protocol described below.

Analytical method: Analyze sample stability by the method listed in Table 1

Table 1: Analytical methods analyze Purpose Color, Transparency, ^Appearancea Visual Inspection of Liquid Dosages Size Exclusion Chromatography (SEC) ^b Measure percentages of monomers, soluble aggregates, and low molecular weight fractions Hydrophobic Interaction Chromatography (HIC) ^c Measurement of Asp-32 isomerization and levels of free thiols UV Spec Scan(Gravimetric) ^f Measuring protein concentration Turbidity (average OD 340-360nm) ^d Measures soluble and insoluble aggregates Active ^e Determining Anti-IgE Binding Activity

^a Check of color, appearance and transparency :

The color, appearance and clarity of the samples were assessed visually against the white and black backgrounds used for inspection, compared to an equal volume of negative controls. The sample should be swirled carefully to ensure homogeneous mixing, but not so vigorously as to generate air bubbles.

^b size exclusion chromatography :

The TSK SUPER SW3000 (4.6×300mm) column was used in the HP 1100 chromatography system. The column was loaded with 20 μg of protein and eluted in 0.1 M potassium phosphate, pH 6.8. Samples were detected with a UV detector at 280 nm.

^c Hydrophobic Interaction Chromatography (HIC) :

HIC experiments were performed on HP 1100 liquid chromatography system using TSK Phenyl-5PW (7.5×75mm) column (TosoHaas). The column was loaded with 28 μg of papain-digested Fab fragments and eluted with a gradient of ammonium sulfate from 2M to 0M in 20 mM Tris buffer. The peak was monitored by a UV detector at 210 nm.

^d Turbidity :

The turbidity of the samples was determined using an HP spectrophotometer in a 1 cm pass length cuvette. Turbidity was calculated as the average absorbance at 340-360 nm.

^e Activity of anti-IgE monoclonal antibodies was determined by receptor binding inhibition assay. Samples were diluted to within the standard curve range of 100 and 1.56 μg/ml in assay diluent containing phosphate buffer, 0.5% BSA, 0.05% polysorbate 20, 0.01% Thimerosol. Microtiter plates were coated with IgE receptors and incubated with IgE-biotin and diluted anti-IgE samples. The amount of receptor-bound IgE-biotin correlated with the activity of anti-IgE monoclonal antibodies was determined using streptavidin-HRP. Data were analyzed using a 4 parameter logistic curve fitting program.

^f Concentrations of antibodies were obtained on a Hewlett Packard 8453 dual column spectrophotometer using 1 cm quartz cuvettes. Concentrations were calculated using an absorbance of 1.5 cm ^-1 (mg/ml) ^-1 .

Summary of liquid preparations preparation protein range buffer/range Excipients/Scope 80mg/ml E25 50mM Histidine-HCl 150mM Trehalose 0.05% Polysorbate 20pH6.0 40-150mg/ml His-HCl or His-acetate range: 10mM-100mM Trehalose or Sucrose Sugar Range: 20mM-350mM Polysorbate: 0.01%-0.1% 150mg/ml E25 20mM Histidine-HCl 200mM ArgHCl 0.02% Polysorbate 20pH6.0 40-260mg/ml His-HCl or His-acetate range: 10mM-100mM ArgHCl range: 50mM-200mM Polysorbate: 0.01%-0.1%

Stability Data of 150mg/ml E25 in Histidine and ArgHCl Formulations temperature(℃) time (month) visual inspection pH SEC ^a monomer% ^HICb %Main Effectiveness ^c Turbidity ^d 5 01316 by by by by by 6.26.06.06.0 99.099.299.398.9 64636362 10610011183 0.250.270.250.27 30 1316 through through through 5.96.16.0 98.4397.5390.63 544219 916528 0.250.300.54

Stability data of 80mg/ml E25 in histidine and trehalose formulations temperature(℃) time (month) visual inspection pH SEC ^a monomer% HIC ^b main component % Effectiveness ^c Turbidity ^d 5 01361424 through through through through through through 5.75.85.75.75.75.7 99.198.798.899.199.098.8 646363636262 10092124978384 0.200.200.200.210.210.20 30 13614 by by by by by 5.85.75.85.7 98.797.495.593.1 55413122 77764830 0.200.290.380.48

a. Measurement of soluble aggregates and fragments by size exclusion chromatography

b. Hydrophobic interaction chromatography on papain-digested E25

c. IgE receptor binding inhibition assay

d. Average OD value (340-360nm)

Stir research: achieve after shaking for 3 days preparation visual inspection SEC (monomer %) Turbidity visual inspection SEC (monomer %) Turbidity 12 Pass 99.5 0.18 Pass 99.0 0.19 Passed 99.3 0.18 Passed 99.4 0.19

Formulation 1: 156mg/ml E25, 200mMArgCl, 23mM His, 0.02% T20

Formulation 2: 150mg/ml E25, 182mMArgCl, 20mM His, 0.02% T20

Freeze-thaw studies: achieve After the 1st cycle After the 3rd cycle preparation visual inspection SEC (monomer %) Turbidity visual inspection SEC (monomer %) Turbidity visual inspection SEC (monomer %) Turbidity 12 Pass 99.5 0.18 Pass 99.0 0.19 Passed 99.3 0.17 Passed 99.2 0.19 through through 99.499.2 0.170.18

Formulation 1: 156mg/ml E25, 200mMArgCl, 23mM His, 0.02% T20

Formulation 2: 150mg/ml E25, 182mMArgCl, 20mM His, 0.02% T20

Example 3

Samples of anti-IgE monoclonal antibody (E26) liquid formulations were prepared in 20 mM buffer and stored at 30°C and 40°C. The stability of E26 was determined by chromatography and activity measurements. Soluble aggregates were determined by size exclusion chromatography and isomerization was measured by hydrophobic interaction chromatography on pepsin digested samples. Samples were monitored for activity using an IgE receptor binding inhibition assay. As shown in Figures 1, 2 and 3, the degradation of E26 is highly dependent on the pH of the buffer. E26 appears to be most stable around pH 6.0.

Example 4

Microparticle formulations present a major challenge in the manufacture of high-concentration liquid formulations, as it typically increases with protein concentration under stress conditions. Figure 4 shows the results of agitation studies on concentrated E26 liquid formulations. Formulations were prepared in 20 mM succinate, 192 mM trehalose, pH 6.0, and various concentrations of polysorbate 20. Microparticle formulations were monitored using turbidity measurements. The results show that the turbidity of the E26 solution increases with agitation time. The addition of at least 0.01% polysorbate under stress is essential to reduce particle formation. Similar results were also observed for the concentrated E25 liquid formulation.

Example 5

Figure 5 shows a liquid formulation of 150 mg/ml E25 prepared by reconstituting lyophilized E25. Increases in salt concentration suppressed reversible particulate formation and resulted in lower turbidity readings. Of all the salts tested, the formulation containing Arg-HCl appeared to have the least turbidity. The effect of salt concentration on reducing turbidity readings was also observed for E25 prepared by the TFF method.

Example 6

The E25 liquid formulation in the presence of ArgHCl also appeared to have better stability than the other liquid formulations. Figures 6 and 7 show the results of a stability study of 150 mg/ml of E25 in a liquid formulation comprising ArgHCl, _CaCl2 and _MgCl2 . For the liquid formulations containing ArgHCl, with or without sucrose, there was little difference in their stability in terms of turbidity, isomerization and fragmentation. Liquid formulations containing ArgHCl were more stable than liquid formulations containing _MgCl2 and _CaCl2 .

Example 7

Figure 8 shows the results of a stability study on the E25 liquid formulations containing acetate and histidine formulations. Formulations containing histidine had a higher pH than acetate formulations. The results clearly show that E25 is more stable in the histidine, ArgHCl liquid formulation than under other conditions.

Example 8

High concentrations of E25 can form solid gels in the presence of certain ions such as citrate, succinate and sulfate (Table 1), especially at storage temperatures of 2-8°C. Utilizing arginine-HCl as an excipient allowed us to formulate E25 to over 200 mg/ml without gel formation or precipitation.

Table 1: Effect of various excipients on the gelation of E25 at 125 mg/ml, pH ~ 6.0 excipient Excipient concentration MM products Turbidity (340-360nm) Antibody concentration mg/ml visual appearance SWFINaCl Succinate Succinate Citrate Citrate Na ₂ SO ₄ Na ₂ SO ₄ Phosphate Acetate Acetate Acetate Histidine Histidine Arginine-HCl Arginine-HCl Arginine Acid - SO4CaCl ₂ MgCl ₂ 188941918819 TBD TBD 18894199447150200150125125 Lyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconLyo ReconTFFLyo ReconTFFTFF 0.210.250.310.28 to be determined 125125125125125125125125125125125125125125137162137147147 Clear Clear Gel Gel Gel Gel Milky Milky Clear Clear Clear Clear Clear Gel Clear Milky

Example 9

Expressing Proteins or Antibodies in E. coli

This example illustrates the production of aglycosylated forms of desired proteins or antibodies by recombinant expression in E. coli.

The DNA sequence encoding the desired protein or antibody is initially amplified using selected PCR primers. Primers should contain restriction enzyme sites corresponding to the restriction enzyme sites on the chosen expression vector. Various expression vectors can be used. An example of a suitable vector is pBR322 (derived from Escherichia coli; see Bolivar et al., Gene, 2:95 (1977)) containing the ampicillin and tetracycline resistance genes. The vector is digested with restriction enzymes and dephosphorylated. The PCR-amplified sequences are then ligated into the vector. The vector preferably includes a sequence encoding an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, a polyhis sequence and an enterokinase cleavage site), the coding region of the desired protein or antibody, and the lambda transcription terminator and argU gene. In addition, the vector may include at least nonsense portions of the untranslated 5' and 3' fragments of the native nucleic acid sequence encoding the desired protein or antibody.

The ligation mixture was then used to transform selected E. coli strains using the method described in Sambrook et al., supra. Transformants were identified by their ability to grow on LB plates, and antibiotic resistant colonies were selected. Plasmid DNA can be isolated, confirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid media, such as LB broth supplemented with antibiotics. Overnight cultures can then be used to inoculate larger cultures. The cells are then grown to the desired optical density at which time the promoter is opened for expression.

After culturing the cells for a few more hours, the cells can be harvested by centrifugation. The cell pellet obtained by centrifugation is lysed with various reagents known in the art, and then the solubilized desired protein or antibody can be purified using a metal chelate column under conditions that allow tightly bound proteins or antibodies.

The desired protein or antibody can be expressed in a poly-His-tagged form in Escherichia coli using the following steps. The DNA sequence encoding the desired protein or antibody is initially amplified using selected PCR primers. Primers contain restriction endonuclease sites corresponding to those on the selected expression vector, and others for efficient and reliable translation initiation, rapid purification on metal-chelating columns, use of enterokinase protein Hydrolysis removes the useful sequence provided. The PCR-amplified, poly-His-tagged sequence was then ligated into an expression vector for transformation of a strain 52-based E. coli host (W3110 fuhA(tonA)lon galE rpoHts(htpRts)clpP(lacIq). First Transformants were cultured with shaking at 30° C. in LB containing 50 mg/ml carbenicillin until reaching an OD600 of 3-5. The culture was then diluted 50-100 times into CRAP medium (by mixing 3.57 g ( NH ₄ ) ₂ SO ₄ , 0.71g sodium citrate $2H2O, 1.07g KCl, 5.36g Difco yeast extract, 5.36g Sheffield hycase SF, and 110mM MPOS, pH7.3, 0.55% (w/v) glucose and 7mM MgSO ₄ ) and grown with shaking at 30°C for approximately 20-30 hours. Samples were taken to confirm expression by SDS-PAGE analysis, and bulk cultures were centrifuged to pellet cells. Cell pellets were frozen until initial purification and Refold.

Resuspend the Escherichia coli (6-10g cell mass) obtained from 0.5 to 1L fermentation broth in 10 times the amount (w/v) of 7M guanidine, 20mM Tris, pH8 buffer. Solid sodium sulfite and sodium tetrathionate were added to a final concentration of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4°C. This step yields a denatured protein in which all cysteine residues are blocked by sulfitation. The solution was centrifuged at 40,000 rpm for 30 minutes in a Beckman Ultracentifuge. Dilute the supernatant with 3-5 volumes of metal chelating column buffer (6M guanidine, 20mM Tris, pH7.4), and filter through a 0.22 micron filter to clarify. The clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade) pH 7.4. Proteins were eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4°C. Protein concentrations were estimated by absorbance at 280 nm using extinction coefficients calculated from their amino acid sequences.

The protein was refolded by slowly diluting the sample into freshly prepared refolding buffer consisting of: 20mM Tris, pH 8.6, 0.3M NaCl, 2.5M urea, 5mM cysteine, Composed of 20mM glycine and 1mM EDTA. The refolding volume is chosen such that the final protein concentration is between 50-100 μg/ml. The refolding solution was stirred gently for 12-36 hours at 4°C. The refolding reaction was terminated by adding TFA to a final concentration of 0.4% (pH ~3). Before further protein purification, the solution was filtered through a 0.22 micron filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was chromatographed on a Poros R1/H reversed-phase column using 0.1% TFA in running buffer with a gradient of acetonitrile from 10% to 80%. Aliquots of fractions with A280 absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded proteins were pooled. In general, the properly refolded species of most proteins elute at the lowest acetonitrile concentration because these folded protein species are the most compact, with their hydrophobic interiors shielded from interaction with the reversed-phase resin. Aggregated species generally elute at higher acetonitrile concentrations. In addition to removing the misfolded form of the protein from the desired form of the protein, the reverse phase step also removes endotoxin from the sample.

Fractions containing the folded desired protein or antibody were collected and the acetonitrile was removed using a gentle stream of nitrogen in solution. Proteins were formulated in 20 mM Hepes, pH 6.8, containing 0.14 M NaCl and 4% mannitol, by dialysis or by gel filtration using G25 Superfine (Pharmacia) resin equilibrated in formulation buffer, and run without Bacterial filtration.

Example 10

Express proteins or antibodies in mammalian cells

This example illustrates the production of potentially glycosylated forms of desired proteins or antibodies by recombinant expression in mammalian cells.

The vector pRK5 (see EP 307,247 published on March 15, 1989) was used as an expression vector. Alternatively, DNA encoding the desired protein or antibody is ligated into pRK5 using restriction enzymes selected to allow insertion of this DNA using ligation methods such as described in Sambrook et al., supra.

In one embodiment, the selected host cell may be a 293 cell. Human 293 cells (ATCCCCL 1573) are grown to confluency on tissue culture plates in medium, such as DMEM supplemented with fetal calf serum and optionally supplemented with nutrients and/or antibiotics. About 10 μg of DNA encoding a desired protein or antibody ligated into pRK5 was mixed with about 1 μg of DNA encoding a VA RNA gene [Thimmappaya et al., Cell, 31 :543 (1982)], and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227M CaCl ₂ . To this mixture was added dropwise 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ and allowed to form a precipitate at 25° C. for 10 minutes. The pellet was suspended, added to 293 cells, and allowed to settle for approximately four hours at 37°C. Aspirate medium and add 2 ml of 20% glycerol in PBS for 30 sec. The 293 cells were then washed with serum-free medium, fresh medium was added, and the cells were incubated for about 5 days.

Approximately 24 hours after transfection, medium was removed and replaced with medium (alone) or medium containing 200 μCi/ml ³⁵ S-cysteine and 200 μCi/ml ³⁵ S-methionine. After 12 hours of incubation, conditioned medium was collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel can be dried and exposed to film for a selected period of time to reveal the presence of the desired protein or antibody. Cultures containing transfected cells can undergo further incubation (in serum-free medium), testing the medium in selected bioassay methods.

In an alternative technique, the desired protein or antibody can be transiently introduced into 293 cells using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci. 12 :7575 (1981). 293 cells were grown to maximum density in spinner flasks and 700 μg of DNA encoding the desired protein or antibody was added ligated into pRK5. Cells were first concentrated from spinner flasks by centrifugation and washed with PBS. The DNA-dextran pellet was incubated on the cell pellet for four hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and added back to the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned medium was centrifuged and filtered to remove cells and debris. The sample containing the expressed desired protein or antibody can then be concentrated and purified by any method of choice, such as dialysis and/or column chromatography.

In another embodiment, the desired protein or antibody can be expressed in CHO cells. DNA encoding a desired protein or antibody linked to pRK5 can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE-dextran. As described above, cell cultures may be incubated and medium replaced by medium (alone) or medium comprising a radioactive label such as 35S-methionine. After determining the presence of the desired protein or antibody, the medium can be replaced with serum-free medium. Preferably, the culture is incubated for about 6 days before harvesting the conditioned medium. The medium containing the expressed desired protein or antibody can then be concentrated and purified by any method of choice.

Epitope-tagged variants of the desired protein or antibody can also be expressed in host CHO cells. DNA encoding a desired protein or antibody ligated into pRK5 can be subcloned from the pRK5 vector. Subcloned inserts can be PCR fused in-frame with a selected epitope tag, such as a poly-his tag, into a baculovirus expression vector. The poly-his tagged DNA encoding the desired protein or antibody insert can then be subcloned into an SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, CHO cells can be transfected (as described above) with the SV40 driven vector. Expression can be confirmed by labeling as described above. The medium containing the expressed poly-His tagged desired protein or antibody can then be concentrated and purified by any method of choice, such as Ni ²⁺ chelate affinity chromatography.

Desired proteins or antibodies can also be expressed in CHO and/or COS cells by a transient expression step, or in CHO cells by another stable expression step.

Stable expression in CHO cells was performed using the following procedure. Proteins are expressed as IgG constructs (immunoadhesins) in which the coding sequence of the soluble form (e.g., extracellular region) of the respective protein is fused to IgG1 constant region sequences comprising the hinge region, CH2 and CH2 regions, and/or is a poly-His tagged form.

Following PCR amplification, each DNA was subcloned into a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). The CHO expression vector is constructed with restriction sites 5= and 3= compatible with the DNA of interest to allow easy movement of the cDNA. The vector used in CHO cell expression used the SV40 early promoter/enhancer to drive the cDNA of interest and dihydrofolate reduction as described in Lucas et al., Nucl. Acids Res. 24 :9 (1774-1779 (1996). Expression of Enzyme (DHFR). DHFR expression allows selection of cells stably maintaining the plasmid after transfection.

Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect ^™ (Quiagen), Dosper ^™ or Fugene ^™ (Boehringer Mannheim). Cells were grown as described in Lucas et al., supra. Approximately 3 x 10 ^-7 cells were frozen in ampoules for further growth and production as described below.

Ampoules containing plasmid DNA were thawed by placing in a water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mL of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selection medium (0.2 m filtered PS20 containing 5% 0.2 m filtered fetal bovine serum). Cells were then aliquoted into 100 mL rotators containing 90 mL selection medium. After 1-2 days, cells were transferred to a 250 mL rotator filled with 150 mL of selective growth medium and incubated at 37°C. After another 2-3 days, inoculate 250 mL, 500 mL and 2000 mL spinners at 3 x ¹⁰⁵ cells/mL. Cell culture medium was replaced with fresh medium by centrifugation and resuspension in production medium. Although any suitable CHO medium can be used, in practice the production medium described in US Patent No. 5,122,469 issued June 16, 1992 can be used. Inoculate a 3 L production spinner at 1.2 x ¹⁰⁶ cells/mL. On day 0, cell number and pH were determined. On day 1, the rotator was sampled and aeration with filtered air was initiated. On day 2, sample the rotator, change temperature to 33°C, add 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (eg, 35% dimethicone emulsion, Dow Coming 365 medical grade emulsion) . During the whole production process, the pH is adjusted as needed and kept around 7.2. After 10 days, or until the viability dropped below 70%, the cell cultures were harvested by centrifugation and filtration through a 0.22 <RTI ID=0.0>Fm</RTI> filter. The filtrate can be stored at 4°C, or immediately loaded onto a column for purification.

For poly-His tagged constructs, proteins were purified using Ni-NTA columns (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned medium was pumped at a flow rate of 4-5 ml/min onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4 buffer containing 0.3 M NaCl and 5 mM imidazole. After loading, the column was washed with additional equilibration buffer and proteins were eluted with equilibration buffer containing 0.25M imidazole. The highly purified protein was then desalted with a 25ml G25Superfine (Pharmacia) column, placed in a storage buffer containing 10Hepes, 0.14MNaCl and 4% mannitol, pH6.8, and stored at -80°C.

Immunoadhesin (Fc containing) constructs were purified from conditioned medium as described below. The conditioned medium was pumped onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM Na phosphate buffer, pH 6.8. After loading, the column was carefully washed with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. Immediately neutralize the eluted protein by collecting 1 ml fractions into tubes containing 275 L 1M Tris buffer, pH 9. The highly purified protein was then desalted and placed into storage buffer as described above for poly-His protein. Homogeneity was assessed by SDS polyacrylamide gels and N-terminal amino acid sequencing by Edman degradation.

Example 11

Expressing Antibodies or Proteins in Yeast

The following methods describe the recombinant expression of a desired protein or antibody in yeast.

First, a yeast expression vector is constructed for intracellular production or secretion of the desired protein or antibody from the ADH2/GAPDH promoter. The DNA encoding the desired protein or antibody and the promoter are inserted into the appropriate restriction enzyme sites in the selected plasmid for direct intracellular expression. For secretion, together with DNA encoding the ADH2/GAPDH promoter, native signal peptide or other mammalian signal peptide, or, for example, yeast alpha factor or invertase secretion signal/leader sequence, and linker sequences (if desired), the encoding The DNA of the desired protein or antibody is cloned into a selected plasmid for expression of the desired protein or antibody.

Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and grown in a selected fermentation medium. Analysis can be performed by precipitation of transformed yeast supernatants with 10% trichloroacetic acid, separation by SDS-PAGE, and staining of the gel with Coomassie blue.

Yeast cells are subsequently removed from the fermentation medium by centrifugation, followed by concentration of the medium with selected cartridge filters for isolation and purification of recombinant proteins or antibodies. Concentrates containing recombinant proteins or antibodies can be further purified using selected column chromatography resins.

Example 12

Expression of proteins or antibodies in baculovirus-infected insect cells

The following methods describe recombinant expression of desired proteins or antibodies in baculovirus-infected insect cells.

A sequence encoding the desired protein or antibody is fused upstream of the epitope tag contained in the baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like the Fc region of IgG). Various plasmids can be used, including those derived from commercially available ones, such as pVL1393 (Novagen). Briefly, sequences encoding the desired portion of the protein or antibody are amplified by PCR using primers complementary to the 5' and 3' regions, for example, sequences encoding the extracellular region of a transmembrane protein, or if the protein is an extracellular protein, Sequence encoding the mature protein. The 5' primer may include flanking (selected) restriction enzyme sites. The products are then digested with those selected restriction enzymes and subcloned into expression vectors.

Recombinant baculoviruses were generated by co-transfecting the above plasmids and BaculoGold ^™ viral DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCCCRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28°C, the released virus was harvested for further amplification. Virus infection and protein expression were performed as described in O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

The expressed poly-his-tagged protein or antibody can then be purified by, for example, Ni ²⁺ chelate affinity chromatography as follows. Extracts were prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362 : 175-179 (1993). Briefly, Sf9 cells were washed and resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4M KCl) on ice Sonicate twice for 20 seconds. The sonicate was clarified by centrifugation, and the supernatant was diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 m filter. A Ni ²⁺ -NTA agarose column (commercially available from Qiagen) was prepared with a bed volume of 5 mL, washed with 25 mL of water, and equilibrated with 25 mL of loading buffer. The filtered cell extract was loaded onto the column at 0.5 mL per minute. The column was washed with loading buffer to baseline _A280 , at which point fraction collection was initiated. Then, the column was washed with a second wash buffer (50 mM phosphate; 300 mM NaCI, 10% glycerol, pH 6.0) to elute non-specifically bound proteins. After reaching the A280 baseline again, the column was run with a gradient of 0 to 500 mM imidazole in the second wash buffer. One ml fractions were collected and analyzed by SDS-PAGE and silver staining or Western blotting with Ni2 ⁺ -NTA conjugated to alkaline phosphatase (Qiagen). Fractions containing eluted His ₁₀ -tagged protein or antibody were pooled and dialyzed against loading buffer.

Alternatively, purification of IgG-tagged (or Fc-tagged) proteins or antibodies can be performed using known chromatographic techniques including, for example, protein A or protein G column chromatography.

Example 13

Antibody preparation

This example illustrates the preparation of monoclonal antibodies that bind a protein of interest or a desired antigen.

Techniques for producing monoclonal antibodies are known in the art, eg, described in Goding, supra. Immunogens that can be used include purified desired protein or antibody of interest, fusion proteins comprising the desired protein or antigen of interest, and cells expressing such recombinant protein or antigen on the cell surface. Immunogens can be selected by the skilled artisan without undue experimentation.

Mice, eg Balb/c, are immunized with the desired protein or immunogen of interest emulsified in complete Freund's adjuvant, injected subcutaneously or intraperitoneally in an amount of 1-100 micrograms. Alternatively, the immunogen was emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind footpad. Immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. The mice may also be boosted with additional immunization injections for several weeks thereafter. Serum samples were periodically obtained from mice by retro-orbital bleeding for detection of antibodies against desired proteins or antigens in ELISA assays.

Animals "positive" for antibodies may be given a final intravenous injection with the desired protein or target antigen after detection of suitable antibody titers. Three to four days later, mice were sacrificed and splenocytes were harvested. The splenocytes are then fused (using 35% polyethylene glycol) with a selected murine myeloma cell line, such as P3X63AgU.1 available from ATCC, No. CRL 1597. Fusion produces hybridoma cells, which can then be plated in 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to suppress non-fused cells, myeloma hybrids and proliferation of splenocyte hybrids.

ELISA is used to screen hybridoma cells for reactivity to desired proteins or target antigens. It is within the skill of the art to determine "positive" hybridoma cells secreting such monoclonal antibodies.

The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to generate ascites fluid containing such monoclonal antibodies. Alternatively, hybridoma cells can be grown in tissue culture flasks or roller bottles. Monoclonal antibodies produced in peritoneal fluid can be purified using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on antibody binding to protein A or protein G can be used.

Example 14

Purify desired protein using specific antibody

A desired protein in native or recombinant form can be purified by various standard techniques in the protein purification art. For example, a precursor polypeptide, mature polypeptide, or propolypeptide form of a desired protein can be purified by immunoaffinity chromatography using antibodies specific for the desired protein. Generally, immunoaffinity chromatography columns are constructed by covalently linking antibodies that specifically bind the desired protein to activated chromatography resins.

Polyclonal immunoglobulins were prepared from immune sera by precipitation with ammonium sulfate or by purification on immobilized protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Likewise, monoclonal antibodies were prepared from mouse peritoneal fluid by ammonium sulfate precipitation or chromatography on immobilized protein A. Partially purified immunoglobulins are covalently attached to a chromatography resin, such as CnBr-activated SEPHAROSE ^™ (Pharmacia LKB Biotechnology). Antibody was coupled to the resin, resin blocked, and derivatized resin washed according to the manufacturer's instructions.

This immunoaffinity column is used to purify the desired protein by preparing fractions from cells expressing the protein in soluble form. Such preparations are obtained by lysing whole cells or subcellular fractions obtained by differential centrifugation by addition of detergent or other methods known in the art. Alternatively, the soluble protein comprising the signal sequence can be secreted into the medium in which the cells are grown in an effective amount.

The solubilized preparation containing the desired protein is passed through the immunoaffinity column, and the column is washed under conditions that allow preferential uptake of the desired protein (eg, high ionic strength buffer in the presence of detergent). The column is then washed in conditions that disrupt the binding of the antibody to the protein (e.g., a low pH buffer, such as about pH 2-3, or a high concentration of a chaotrope, such as urea or thiocyanate ions), to collect the desired protein.

Claims (50)

1. the liquid preparation of stable, a low turbidity, the albumen or the antibody that comprise (a) 100 to 260mg/ml amounts, (b) arginine-HCl of 50 to 200mM amounts, (c) histidine of 10 to 100mM amounts, (d) polysorbate of 0.01 to 0.1% amount, wherein said preparation further have from 5.5 to 7.0 pH value, about 50cs or littler kinematic viscosity and the osmotic pressure from 200mOsm/kg to 450mOsm/kg.

2. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 120mg/ml to 260mg/ml.

3. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 150mg/ml to 260mg/ml.

4. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 180mg/ml to 260mg/ml.

5. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 200mg/ml to 260mg/ml.

6. the described preparation of claim 1, the about 150mg/ml of the concentration of wherein said albumen or antibody.

7. the described preparation of claim 1, wherein said osmotic pressure scope is from 250mOsm/kg to 350mOsm/kg.

8. the described preparation of claim 1, wherein the concentration range of arginine-HCl is from 100mg/ml to 200mg/ml.

9. the liquid preparation of stable, a low turbidity, the anti-IgE Mab that comprises (a) 100 to 260mg/ml amounts, (b) arginine-HCl of 50 to 200mM amounts, (c) histidine of 10 to 100mM amounts, (d) polysorbate of 0.01 to 0.1% amount, wherein said preparation further have from 5.5 to 7.0 pH value, about 50cs or littler kinematic viscosity and the osmotic pressure from 200mOsm/kg to 450mOsm/kg.

10. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 120mg/ml to 260mg/ml.

11. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 150mg/ml to 260mg/ml.

12. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 180mg/ml to 260mg/ml.

13. the described preparation of claim 1, the concentration range of wherein said albumen or antibody is from 200mg/ml to 260mg/ml.

14. the described preparation of claim 1, the about 150mg/ml of the concentration of wherein said albumen or antibody.

15. the described preparation of claim 1, the scope of wherein said osmotic pressure is from 250mOsm/kg to 350mOsm/kg.

16. the described preparation of claim 1, wherein said anti-IgE antibodies is selected from rhuMAbE25, rhuMAbE26 or Hu-901.

17. the described preparation of claim 1, wherein said anti-IgE antibodies is rhuMAbE25.

18. the described preparation of claim 1, wherein said anti-IgE antibodies is rhuMAbE26.

19. the described preparation of claim 1, wherein said anti-IgE antibodies is Hu-901.

20. liquid preparation stable, low turbidity, comprise the anti-IgE antibodies of the about 150mg/ml of (a) amount, (b) amount is arginine-HCl of 200mM, and (c) amount is the histidine of 20mM, (d) amount is 0.02% polysorbate, and wherein said preparation further has 6.0 pH value.

21. the described preparation of claim 20, wherein said anti-IgE antibodies is E25.

22. goods comprise the container that the described preparation of claim 1 is housed.

23. the described goods of claim 22, wherein said container is a syringe.

24. the described goods of claim 23, wherein said syringe further is comprised in the injection device.

25. the described goods of claim 24, wherein said injection device is an automatic injector.

26. the described preparation of claim 1, wherein said preparation is reconstruct.

27. the described preparation of claim 26, wherein doubly at the about high 2-40 of the concentration of concentration ratio before lyophilizing of albumen described in the preparation of described reconstruct or antibody.

28. cure the method for the disease of IgE mediation, comprise the preparation of the claim 20 of patient's administering therapeutic effective dose of curing to needs.

29. the described method of claim 28, the disease of wherein said IgE mediation is selected from allergic rhinitis, asthma, allergic asthma, non-allergic asthma, atopic dermatitis or gastrointestinal disease.

30. the described method of claim 28, the disease of wherein said IgE-mediation is an allergic rhinitis.

31. the described method of claim 28, the disease of wherein said IgE-mediation is an allergic asthma.

32. the described method of claim 28, the disease of wherein said IgE-mediation is an asthma.

33. the described method of claim 28, the disease of wherein said IgE-mediation is an atopic dermatitis.

34. the described method of claim 28, the disease of wherein said IgE mediation is selected from allergy, allergic bronchopulmonary aspergillosis, parasitic disease, interstitial cystitis, high IgE syndrome, ataxia telangiectasia, Wiskott-Akdrich syndrome, thymic alymphoplasia, IgE myeloma or graft versus host disease.

35. the described method of claim 28, the disease of wherein said IgE mediation is allergy.

36. the described method of claim 35, wherein said allergy disease is selected from anaphylaxis, urticaria or food anaphylaxis.

37. the described method of claim 36, wherein allergy disease are food anaphylaxiss.

38. the described method of claim 37, wherein said food anaphylaxis are by to the exposure of beans and cause.

39. the described method of claim 38, wherein said beans is a Semen arachidis hypogaeae.

40. cure the method for the disease of IgE mediation, comprise the preparation of patient's effective dosage and the claim of curing to the needs 20 hydryllin combination.

41. cure the method for the disease of IgE mediation, comprise antihistaminic the using of associating, the preparation of the claim 20 of patient's drug treatment effective dose of curing to needs.

42. cure the method for the disease of IgE mediation, comprise the preparation of patient's drug treatment effective dose and the claim of curing to the needs 20 bronchodilators combination.

43. cure the method for the disease of IgE mediation, comprise using of associating bronchodilators, to the preparation of the claim 20 of patient's drug treatment effective dose of needs treatment.

44. cure the method for the disease of IgE mediation, comprise the preparation of patient's drug treatment effective dose and the claim of curing to the needs 20 glucocorticoid combination.

45. cure the method for the disease of IgE mediation, comprise using of combination with glucocorticoids, the preparation of the claim 20 of patient's drug treatment effective dose of curing to needs.

46. cure the method for the disease of IgE mediation, comprise the preparation of the claim 20 of the treatment effective dose that patient's administration of curing to needs and glucocorticoid make up.

47. cure the method for the disease of IgE mediation, comprise using of combination with glucocorticoids, the preparation of the claim 20 of patient's drug treatment effective dose of curing to needs.

48. cure the method for the disease of IgE mediation, comprise using of associating allergen desensitization therapy, to the preparation of the claim 20 of patient's drug treatment effective dose of needs treatment.

49. cure the method for the disease of IgE mediation, comprise the preparation of patient's drug treatment effective dose and the claim of curing to the needs 20 NSAID combination.

50. cure the method for the disease of IgE mediation, comprise using of associating NSAID, to the preparation of the claim 20 of patient's administering therapeutic effective dose of needs treatment.

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